Methods for detecting pre-diabetes and diabetes using differential protein glycosylation

ABSTRACT

Methods for identifying individuals who are not yet diabetic (pre-diabetic), but who are at significant risk of developing diabetes, such as type 2 diabetes, are disclosed herein. Methods are also provided for the identification of diabetic subjects. Also disclosed are methods for identifying individuals with diabetic complications. The methods include the identification of an overall glycosylation profile of proteins in a biological fluid, such as saliva, urine, or serum. In some examples, the methods include determining the amount of one or more protein in a biological fluid or determining the glycosylation pattern of one or more proteins in a biological fluid.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of and claims priority to U.S.patent application Ser. No. 12/777,955, filed May 11, 2010, entitled“METHODS FOR DETECTING PRE-DIABETES AND DIABETES USING DIFFERENTIALPROTEIN GLYCOSYLATION,” which claims priority to U.S. Provisional PatentApplication No. 61/177,130, filed May 11, 2009, entitled “METHODS FORDETECTING PRE-DIABETES AND DIABETES USING DIFFERENTIAL PROTEINGLYCOSYLATION,” the disclosures of which are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

This relates to the field of diabetes, specifically to theidentification of subjects who have pre-diabetes, diabetes, or diabeticcomplications.

BACKGROUND

Diabetes mellitus is a metabolic disorder characterized by chronichyperglycemia with disturbances of carbohydrate, fat, and proteinmetabolism that result from defects in insulin secretion, insulinaction, or both. Diabetes can present with characteristic symptoms suchas thirst, polyuria, blurring of vision, chronic infections, slow woundhealing, and weight loss. In its most severe forms, ketoacidosis or anon-ketotic hyperosmolar state may develop and lead to stupor, coma,and, in the absence of effective treatment, death.

Diabetes mellitus is subdivided into type 1 diabetes and type 2diabetes. Type 1 diabetes (T1DM) results from auto-immune mediateddestruction of the beta cells of the pancreas. Patients with T1DMexhibit little or no insulin secretion as manifested by low orundetectable levels of insulin or plasma C-peptide (also known in theart as “soluble C-peptide”). Type 2 diabetes (T2DM) is characterized bydisorders of insulin action and insulin secretion, either of which maybe the predominant feature. T2DM patients can be both insulin deficientand insulin resistant. At least initially, and often throughout theirlifetime, these individuals do not need supplemental insulin treatmentto survive. T2DM accounts for 90-95% of all cases of diabetes and can goundiagnosed for many years because the hyperglycemia is often not severeenough to provoke noticeable symptoms of diabetes or symptoms are simplynot recognized. The majority of patients with T2DM are obese, andobesity itself may cause or aggravate insulin resistance. Many of thosewho are not obese by traditional weight criteria may have an increasedpercentage of body fat distributed predominantly in the abdominal region(visceral fat).

The symptoms of the early stages of diabetes often are not severe, notrecognized, or may be absent. Consequently, hyperglycemia sufficient tocause pathological and functional changes may be present for a longtime, occasionally up to ten years, before a diagnosis is made, usuallyby the detection of high levels of glucose in urine after overnightfasting during a routine medical work-up. The long-term effects ofdiabetes include progressive development of complications such asretinopathy with potential blindness, nephropathy that may lead to renalfailure, neuropathy, microvascular changes, and autonomic dysfunction.People with diabetes are also at increased risk of cardiovascular,peripheral vascular, and cerebrovascular disease, as well as anincreased risk of cancer. Several pathogenic processes are involved inthe development of diabetes, including processes that destroy theinsulin-secreting beta cells of the pancreas with consequent insulindeficiency, and changes in liver and smooth muscle cells that result inresistance to insulin uptake. The abnormalities of carbohydrate, fat,and protein metabolism are due to deficient action of insulin on targettissues resulting from insensitivity to insulin (insulin resistance) orlack of insulin (loss of beta cell function).

Over 18 million people in the United States have T2DM, and of these,about 5 million do not know they have the disease. These persons, who donot know they have the disease and who do not exhibit the classicsymptoms of diabetes, present a major diagnostic and therapeuticchallenge. Nearly 41 million persons in the United States are atsignificant risk of developing T2DM. These persons are broadly referredto as “pre-diabetic.” As intervention early in the development ofdiabetes can substantially affect the long-term prognosis of thedisease, it is beneficial to identify individuals who are pre-diabetic,or those subjects who will become diabetic.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will be readily understood by the following detaileddescription in conjunction with the accompanying drawings. Embodimentsare illustrated by way of example and not by way of limitation in thefigures of the accompanying drawings.

FIG. 1A is a perspective view of an example of a lateral flow test stripshowing the basic components of the device and their relationship toeach other, in accordance with various embodiments;

FIG. 1B is a perspective view of an example of a lateral flow deviceshowing the test strip enclosed in a housing, in accordance with variousembodiments;

FIG. 1C shows a perspective view of an example of a lateral flow teststrip for the detection of multiple analytes, in accordance with variousembodiments;

FIG. 2 is a series of digital images showing two-dimensional differencegel electrophoresis of serum glycoproteins from patients with impairedglucose tolerance (IGT; FIG. 2A), impaired fasting glucose (IFG; FIG.2B), type 2 diabetes mellitus (T2DM; FIG. 2C), or T2DM (FIG. 2D)following treatment labeled with Cy5 (patient) or Cy3 (control) dyes, inaccordance with various embodiments;

FIG. 3 is a series of digital images showing two-dimensional differencegel electrophoresis of saliva glycoproteins from patients with impairedglucose tolerance (IGT; FIG. 3A), impaired fasting glucose (IFG; FIG.3B), type 2 diabetes mellitus (T2DM; FIG. 3C), or type 1 diabetesmellitus (T1DM; FIG. 3D) labeled with Cy5 (patient) or Cy3 (control)dyes, in accordance with various embodiments;

FIG. 4 is a graph of mean log-transformed AAL binding in urine fromcontrol, pre-diabetes, and diabetes subjects, in accordance with variousembodiments; and

FIG. 5 is a perspective view of an example of a lateral flow test stripfor diagnosing pre-diabetes or diabetes, in accordance with variousembodiments.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings which form a part hereof, and in which are shownby way of illustration embodiments that may be practiced. It is to beunderstood that other embodiments may be utilized and structural orlogical changes may be made without departing from the scope. Therefore,the following detailed description is not to be taken in a limitingsense, and the scope of embodiments is defined by the appended claimsand their equivalents.

Various operations may be described as multiple discrete operations inturn, in a manner that may be helpful in understanding embodiments;however, the order of description should not be construed to imply thatthese operations are order dependent.

The description may use perspective-based descriptions such as up/down,back/front, and top/bottom. Such descriptions are merely used tofacilitate the discussion and are not intended to restrict theapplication of disclosed embodiments.

The terms “coupled” and “connected,” along with their derivatives, maybe used. It should be understood that these terms are not intended assynonyms for each other. Rather, in particular embodiments, “connected”may be used to indicate that two or more elements are in direct physicalor electrical contact with each other. “Coupled” may mean that two ormore elements are in direct physical or electrical contact. However,“coupled” may also mean that two or more elements are not in directcontact with each other, but yet still cooperate or interact with eachother.

For the purposes of the description, a phrase in the form “NB” or in theform “A and/or B” means (A), (B), or (A and B). For the purposes of thedescription, a phrase in the form “at least one of A, B, and C” means(A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C). For thepurposes of the description, a phrase in the form “(A)B” means (B) or(AB) that is, A is an optional element.

The description may use the terms “embodiment” or “embodiments,” whichmay each refer to one or more of the same or different embodiments.Furthermore, the terms “comprising,” “including,” “having,” and thelike, as used with respect to embodiments, are synonymous.

I. Introduction

The current screening for pre-diabetes includes measuring blood glucoselevels during fasting conditions (to detect impaired fasting glucose;IFG) or following a glucose challenge (to determine impaired glucosetolerance; IGT). These tests aid in the assessment of potential risk fordevelopment of frank diabetes, such as type 2 diabetes mellitus (T2DM),and facilitates the institution of interventions to slow or preventdisease progression. The definition of IGT as a pre-diabetic conditionwas introduced many years ago to define disease risk based on oralglucose tolerance tests (OGTTs). Subsequently, the classification of IFGwas introduced to provide a less complicated and less expensiveparameter. IFG and IGT are now thought to reflect different aspects ofthe development of insulin resistance in T2DM, with differing underlyingpathologies (Abdul-Ghani et al., Diabetes Care 29:1130-1139, 2006).Specifically, IGT is associated with peripheral insulin resistance andloss of first- and second-phase insulin secretion, while IFG isassociated with hepatic insulin resistance and absence of first-phaseinsulin secretion. These distinctions in location of insulin resistanceand extent of beta-cell dysfunction are consistently seen in NativeAmerican (Meyer et al., Diabetes Care 29:1909-1914, 2006),Mexican-American (Abdul-Ghani et al., Diabetes 55:1430-1435, 2006), andadult (Laakso et al., Diabetologia 51:502-511, 2008) and adolescent(Cali et al., J. Clin. Endocrinol. Metab. 93:1767-1773, 2008) Caucasianpopulations.

However, the use of blood glucose levels as a marker of pre-diabetes orfrank diabetes has come under recent scrutiny. Specifically, thepopulation-based studies upon which the current diagnostic criteria arebased suffered from small sample size, inadequate knowledge of theglycemic history of the participants, and the inherently poorreproducibility of OGTTs (Davidson, Curr. Opin. Endocrin. Diabet.12:437-443, 2005; Wong et al., Lancet 371:736-743, 2008). These findingsraise serious questions about the adequacy and relevance of currentdiagnostic criteria for the diagnosis of T2DM. A separate, but relatedissue is the consistency of plasma glucose measurements themselves(Gambino, Clin. Chem. 53:2040-2041, 2007). Thus, these means ofassessing pre-diabetes as well as overt T2DM are fraught with bothtechnical and patient-variability issues.

Although levels of urinary glucose are also unreliable in assessingdiabetic status, the evaluation of increased levels of protein in urineas well as detection of specific urinary proteins is useful fordiagnosis of diabetic nephropathy and other diseases. More in-depthproteomic analysis of urine is expanding the list of possible urinarybiomarkers of disease. Among the urine proteins identified byproteomics, glycoproteins constitute the largest fraction of the urineproteome characterized to date (Adachi et al., Genome Biol. 7:R80, 2006;Sun et al., Proteomics 5:4994-5001, 2005; Wang et al., Mol. Cell.Proteomics 5:560-562, 2006). The glycoproteome has been analyzed usingConcanavalin A-captured glycoproteins from urine samples in healthysubjects and those with glomerular disease (Wang et al., Mol. Cell.Proteomics 5:560-562, 2006; Wang et al., Biochem. Biophys. Res. Comm.371:385-390, 3008). Glycoprotein biomarkers from cells and biologicalfluids such as serum and urine have been further characterized byanalysis of the attached carbohydrate moieties in order to find a morespecific or easily detectable molecular indicator. Examples includeprostate specific antigen (Tabares et al., Glycobiol. 16:132-145, 2006),prion protein species (Pan et al., J. Clin. Microbiol. 43:1118-1126,2005), fibronectin in rheumatoid synovial fluid (Przybysz et al.,Glycoconj. J. 24:543-550, 2007), and alpha-1 acid glycoprotein (A1AG) inamniotic fluid (Orczyk-Pawilowicz et al., Clin. Chem. Acta 367:86-92,2006).

Presented herein in various embodiments are direct lectin assays thatmay be used to detect changes in the glycosylation of biomolecules(glycosylation profile). In embodiments, changes in a glycosylationprofile (such as glycosylation detected by one or more of PHA-E, LEL,DSL, ConA, AAL, or SNA) of a sample and/or a glycosylation pattern ofone or more specific proteins (such as A1AG and A1AT) may be used toidentify a subject as a pre-diabetic or a diabetic or as having adiabetic complication (such as diabetic nephropathy). In some examples,the subjects being tested may be diagnosed with IFG and/or IGT (comparedto non-diabetic controls) and/or may have newly diagnosed type-2diabetes. However, the method also may be used in detecting pre-diabetesor diabetes in subjects who have not been diagnosed with IFG and/or IGT.

Also presented herein in various embodiments are assays that may be usedto determine the expression level and/or glycosylation pattern ofspecific proteins (such as A1AG or A1AT). The differences in theglycosylation profile of a plurality of biomolecules, and/or theexpression or glycosylation pattern or amounts of specific proteinsbetween these conditions may be used in the diagnosis of pre-diabetesand/or diabetes. In embodiments, devices that may be used for measuringthe glycosylation profile are also disclosed.

II. Terms and Abbreviations

-   -   A1AG: alpha-1 acid glycoprotein    -   A1AT: alpha-1 antitrypsin    -   ALA: Aleuria aurantia lectin    -   ConA: Concanavalin A    -   DSL: Datura stramonium lectin    -   ECL: Erythrina cristagalli lectin    -   ELISA: enzyme-linked immunosorbent assay    -   GalNAc: N-acetyl galactosamine    -   GlcNAc: N-acetyl glucosamine    -   GSL-2: Griffonia simplicifolia lectin II    -   HHL: Hippeastrum hybrid lectin    -   IFG: impaired fasting glucose    -   IGT: impaired glucose tolerance    -   LacNAc: N-acetyllactosamine    -   LEL: Lycopersicon esculentum lectin    -   LTL: Lotus tetragonolobus lectin    -   MAL: Maackia amurensis lectin I    -   LEL: Lycopersicon esculentum lectin    -   LTL: Lotus tetragonolobus lectin    -   NDM: newly diagnosed type-2 diabetes mellitus    -   NeuNAc: N-acetylneuraminic acid    -   OGTT: oral glucose tolerance test    -   PHA-E: Phaseolus vulgaris Agglutinin    -   SNA: Sambucus nigra lectin    -   T2DM: type 2 diabetes    -   VVL: Vicia villosa lectin

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). Definitions and additionalinformation known to one of skill in the art in immunology may be found,for example, in Fundamental Immunology, W. E. Paul, ed., fourth edition,Lippincott-Raven Publishers, 1999.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Alpha-1 acid glycoprotein (A1AG): An approximately 41-43 kDa proteinwhich is one of the major acute phase proteins in humans, rats, mice andother species (also known as orosomucoid). The serum concentration ofA1AG increases in response to systemic tissue injury, inflammation orinfection, and is increased in renal complications (such asnephropathy). A1AG has five N-linked glycosylation sites of di-, tri-,and tetraantennary types, with at least twelve glycoforms detected inhuman plasma (Van Dijk et al., Glyconj. J. 12:227-233, 1995).

A1AG sequences are publicly available. For example, GenBank Accessionnumber NC_(—)000009.10 (116125157 . . . 116128578) discloses anexemplary human A1AG type 1 gene sequence (incorporated by reference asincluded in GenBank on May 11, 2009). GenBank Accession numberNM_(—)000607.2 and NP_(—)000598.2 disclose exemplary human A1AG type 1cDNA and protein sequences, respectively (both incorporated by referenceas included in GenBank on May 11, 2009). GenBank Accession numberNC_(—)000009.10 (116131890 . . . 116135357) discloses an exemplary humanA1AG type 2 gene sequence (incorporated by reference as included inGenBank on May 11, 2009). GenBank Accession number NM_(—)000608.2 andNP_(—)000599.1 disclose exemplary human A1AG type 2 cDNA and proteinsequences, respectively (both incorporated by reference as included inGenBank on May 11, 2009). One skilled in the art will appreciate thatA1AG nucleic acid and protein molecules may vary from those publiclyavailable, such as A1AG sequences having one or more substitutions (forexample conservative substitutions), deletions, insertions, orcombinations thereof, while still retaining A1AG biological activity. Inaddition, A1AG molecules include fragments that retain the desired A1AGbiological activity.

Alpha-1 antitrypsin (A1AT): Also known as alpha-1 proteinase inhibitoror Serpin A1. A 52 kDa serine protease inhibitor that is considered themost prominent serpin. The protein was called “antitrypsin” because ofits ability to covalently bind and irreversibly inactivate the enzymetrypsin in vitro. The term alpha-1 refers to the enzyme's behavior onprotein electrophoresis. There are several “clusters” of proteins inelectrophoresis, the first being albumin, the second being the alpha,the third beta and the fourth gamma (immunoglobulins). The non-albuminproteins are referred to as globulins. The alpha region may be furtherdivided into two sub-regions, termed “1” and “2”. Alpha 1-antitrypsin isthe main enzyme of the alpha-globulin 1 region.

A1AT sequences are publicly available. For example, GenBank Accessionnumber NC_(—)000014.7 discloses an exemplary human A1AT gene sequence(93914451 . . . 93926782) (incorporated by reference as included inGenBank on May 11, 2009). GenBank Accession numbers NM_(—)001002235.2,NM_(—)001002236.2, and NM_(—)000295.4 disclose exemplary human A1AT cDNAsequences (each incorporated by reference as included in GenBank on May11, 2009). GenBank Accession numbers NP_(—)001002235.1,NP_(—)001002236.1, and NP_(—)000286.3 disclose exemplary human A1ATprotein sequences (each incorporated by reference as included in GenBankon May 11, 2009). One skilled in the art will appreciate that A1ATnucleic acid and protein molecules may vary from those publiclyavailable, such as A1AT sequences having one or more substitutions (forexample conservative substitutions), deletions, insertions, orcombinations thereof, while still retaining A1AT biological activity. Inaddition, A1AT molecules include fragments that retain the desired A1ATbiological activity.

Analyte: An atom, molecule, group of molecules or compound of natural orsynthetic origin (e.g., glycolipids or glycoproteins). An analyte soughtto be detected or measured that is capable of binding specifically to alectin in some embodiments described herein. Analytes may include, butare not limited to antibodies, drugs, hormones, antigens, haptens,glycoproteins, glycolipids, carbohydrates, apoproteins, or cofactors.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically binds anepitope of a protein listed in the tables below, or a fragment of any ofthese proteins. The term “specifically binds” refers to, with respect toan antigen such the proteins listed in the tables below, thepreferential association of an antibody or other ligand, in whole orpart, with the protein. A specific binding agent binds substantiallyonly to a defined target, such as protein of interest. A minor degree ofnon-specific interaction may occur between a molecule, such as aspecific binding agent, and a non-target polypeptide. Specific bindingmay be distinguished as mediated through specific recognition of theantigen.

A variety of immunoassay formats are appropriate for selectingantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that may be used to determine specific immunoreactivity.

Antibodies may include a heavy chain and a light chain, each of whichhas a variable region, termed the variable heavy (VH) region and thevariable light (VL) region. Together, the VH region and the VL regionare responsible for binding the antigen recognized by the antibody. Thisincludes intact immunoglobulins and the variants and portions of themwell known in the art, such as Fab′ fragments, F(ab)′2 fragments, singlechain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes recombinant forms suchas chimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as, bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies.

Antigen: A chemical or biochemical compound, composition, structure,determinant, protein, glycoprotein or portion thereof that may stimulatethe production of antibodies or a T-cell response in an animal,including compositions that are injected or absorbed into an animal. Anantigen reacts with the products of specific humoral or cellularimmunity, including those induced by heterologous immunogens. The term“antigen” includes all related antigenic epitopes.

Binding affinity: A term that refers to the strength of binding of onemolecule to another at a site on the molecule. If a particular moleculewill bind to or specifically associate with another particular molecule,these two molecules are said to exhibit binding affinity for each other.Binding affinity is related to the association constant and dissociationconstant for a pair of molecules, but it is not critical to theinvention that these constants be measured or determined. Rather,affinities as used herein to describe interactions between molecules ofthe described methods and devices are generally apparent affinities(unless otherwise specified) observed in empirical studies, which may beused to compare the relative strength with which one molecule (e.g., anantibody or other specific binding partner, such as a lectin) will bindtwo other molecules (e.g., an analyte such as a glycoprotein). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

Biological sample: Any biological sample obtained from a plant or animalsubject. As used herein, biological samples include all clinical samplesuseful for detection of glycosylation profile, protein amount, orglycosylation pattern of proteins in subjects, including, but notlimited to, cells, tissues, and bodily fluids, such as: blood;derivatives and fractions of blood, such as serum; extracted galls;biopsied or surgically removed tissue, including tissues that are, forexample, unfixed, frozen, fixed in formalin and/or embedded in paraffin;tears; mucus; saliva; milk; skin scrapes; surface washings; urine;sputum; sweat; semen; vaginal secretion; fluid from ulcers and/or othersurface eruptions, blisters, abscesses, and/or extracts of tissues;cells or organs; cerebrospinal fluid; prostate fluid; pus; or bonemarrow aspirates. The biological sample may also be a laboratoryresearch sample such as a cell culture supernatant. In particularexamples, the sample is urine, saliva, or serum. The sample is collectedor obtained using methods well known to those skilled in the art.

Biomolecule: A molecule that is present in a biological sampleincluding, but not limited to, proteins (such as polypeptides, proteins,or fragments thereof), lipids, and nucleic acids. The term “biomolecule”is specifically intended to cover naturally occurring biomolecules, aswell as those which are recombinantly or synthetically produced. Itshould be noted that the term “biomolecule” includes modified forms ofthe biomolecules, such as glycosylated forms (for example, glycosylatedproteins or peptides or glycosylated lipids).

Capture reagent: An unlabeled specific binding partner that is specificfor (i) an analyte, as in a sandwich assay, or (ii) a detector reagentor an analyte, as in a competitive assay, or for (iii) an ancillaryspecific binding partner, which itself is specific for the analyte, asin an indirect assay. As used herein, an “ancillary specific bindingpartner” is a specific binding partner that binds to the specificbinding partner of an analyte. For example, an ancillary specificbinding partner may include an antibody specific for another antibody,for example, goat anti-human antibody. A “capture area” is a region of alateral flow device where the capture reagent is immobilized. A lateralflow device may have more than one capture area, for example, a “primarycapture area,” a “secondary capture area,” and so on. Often a differentcapture reagent will be immobilized in the primary, secondary, or othercapture areas. Multiple capture areas may have any orientation withrespect to each other on the lateral flow substrate; for example, aprimary capture area may be distal or proximal to a secondary (or other)capture area and vice versa. Alternatively, a primary capture area and asecondary (or other) capture area may be oriented perpendicularly toeach other such that the two (or more) capture areas form a cross or aplus sign or other symbol.

Contacting: “Contacting” includes in solution and solid phase, forexample contacting a salivary sample, urinary sample or protein with atest agent. In one example, contacting includes contacting a sample witha lectin, such as those listed in Table 1 below. In another example,contacting includes contacting a sample with an antibody, for examplecontacting a sample that contains a protein of interest such as alpha-1acid glycoprotein or alpha-1 antitrypsin.

Detecting or Detection: Refers to quantitatively or quantitativelydetermining the presence of the analyte(s) under investigation, such asa glycoprotein.

Detector reagent (or Detection reagent): A specific binding partner thatis conjugated to a label.

Diabetes mellitus: A disease caused by a relative or absolute lack ofinsulin leading to uncontrolled carbohydrate metabolism, commonlysimplified to “diabetes,” though diabetes mellitus should not beconfused with diabetes insipidus. As used herein, “diabetes” refers todiabetes mellitus, unless otherwise indicated. A “diabetic condition”includes pre-diabetes and diabetes. Type 1 diabetes (sometimes referredto as “insulin-dependent diabetes” or “juvenile-onset diabetes”) is anauto-immune disease characterized by destruction of the pancreatic βcells that leads to a total or near total lack of insulin. In type 2diabetes (T2DM; sometimes referred to as “non-insulin-dependentdiabetes” or “adult-onset diabetes”), the body does not respond toinsulin, though it is present. As used herein, the term “metaboliccondition” is used to refer to type 1 diabetes, type 2 diabetes,pre-diabetes, and diabetes complications.

Symptoms of diabetes include: excessive thirst (polydipsia); frequenturination (polyuria); extreme hunger or constant eating (polyphagia);unexplained weight loss; presence of glucose in the urine (glycosuria);tiredness or fatigue; changes in vision; numbness or tingling in theextremities (hands, feet); slow-healing wounds or sores; and abnormallyhigh frequency of infection. Diabetes may be clinically diagnosed by afasting plasma glucose (FPG) concentration of greater than or equal to7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greaterthan or equal to 11.1 mmol/L (200 mg/dL) at about two hours after anoral glucose tolerance test (OGTT) with a 75 g load. A more detaileddescription of diabetes may be found in Cecil Textbook of Medicine, J.B. Wyngaarden, et al., eds. (W.B. Saunders Co., Philadelphia, 1992,19^(th) ed.).

The methods disclosed herein provide a means of identifying s subjectwho has diabetes or pre-diabetes, including both type 1 and type 2diabetes. A “non-diabetic” or “normal” subject does not have any form ofdiabetes, such as type 1 diabetes, type 2 diabetes, or pre-diabetes.

Diabetic complication: Pathologies associated with diabetes that aresecondary to uncontrolled carbohydrate metabolism of diabetes. Acutecomplications (such as hypoglycemia, ketoacidosis, or nonketotichyperosmolar coma) may occur if the blood sugar is not adequatelycontrolled. Chronic elevation of blood glucose level leads to damage ofblood vessels (angiopathy) and chronic complications. In diabetes, theresulting chronic complications are grouped under “microvascularcomplications” (due to damage to small blood vessels) and “macrovascularcomplications” (due to damage to the arteries). Microvascular diabeticcomplications include diabetic nephropathy, diabetic retinopathy,diabetic neuropathy, and diabetic cardiomyopathy. Macrovascularcomplications lead to cardiovascular disease and include coronary arterydisease, stroke, peripheral vascular disease, and diabetic myonecrosis.Additional chronic diabetic complications include diabetic foot, whichresults from a combination of diabetic neuropathy and peripheralvascular disease, and diabetic encephalopathy. In a particular example,the methods disclosed herein include diagnosis of one or more diabeticcomplication, such as microvascular complications, for example, diabeticnephropathy.

Glycosylation: Covalent modification of a biomolecule (such as a proteinor lipid) with one or more oligosaccharide chains. Proteins having atleast one oligosaccharide modification are referred to as“glycoproteins” or “glycosylated proteins.” In the case of proteins,glycosylation is usually N-linked or O-linked. N-linked glycosylationrefers to linkage of an oligosaccharide to the side chain amino group ofan asparagine residue in a protein. O-linked glycosylation refers tolinkage of an oligosaccharide to the hydroxyl side chain of a serine,threonine, or hydroxylysine amino acid in a protein.

The oligosaccharide chains of glycoproteins are enormously varied, dueto the combination of various sugars (for example, N-acetylglucosamine,N-acetylgalactosamine, N-acetyllactosamine, mannose, galactose, glucose,N-acetylneuraminic acid, or fucose) and the presence of branchedstructures (such as biantennary, triantennary, or tetra-antennarystructures).

Glycosylation profile: A representation of the glycosylation (such asthe amount or type of glycosylation) of a plurality of biomolecules(such as a plurality of glycoproteins or glycolipids) in a biologicalsample, such as urine or saliva. The glycosylation profile providesinformation on the amount of one or more type of carbohydrate group (forexample, N-acetylglucosamine, N-acetyl galactosamine, galactose,neuraminic acid, fructose, mannose, fucose, N-acetyllactosamine) orbranch structure (such as bi-, tri-, or tetra-antennary) present on theplurality of biomolecules present in the sample. The glycosylationprofile of a plurality of biomolecules in a sample may be determined bydetecting of binding of biomolecules in the sample to one or morelectins. In some examples, the glycosylation profile of a sample from asubject is compared to the glycosylation profile of a reference in orderto determine whether the subject has pre-diabetes, diabetes, or adiabetic complication.

Glycosylation pattern: The carbohydrate groups attached to a particularbiomolecule (such as a particular glycoprotein or glycolipid), such asthe number, structure, monosaccharide sequence, or location of theindividual sugars on the biomolecule in a biological sample, such asurine or saliva. The glycosylation pattern of a biomolecule may bedetermined by detecting binding of the biomolecule to one or morelectins (such as by lectin-ELISA). In particular examples, theglycosylation pattern of a glycoprotein (such as A1AG or A1AT) in asample from a subject is determined. In some examples, the glycosylationpattern of A1AG and/or A1AT in a sample from a subject is compared tothe glycosylation pattern of A1AG and/or A1AT in a reference in order todetermine whether the subject has pre-diabetes, diabetes, or a diabeticcomplication.

Immunoassay: A biochemical test that measures the presence orconcentration of a substance in a sample, such as a biological sample,using the reaction of an antibody to its cognate antigen, for examplethe specific binding of an antibody to a protein. The presence ofantigen and/or the amount of antigen present may be measured. Formeasuring proteins, for each antigen the presence and amount (abundance)of the protein may be determined or measured. The assay may becompetitive or non-competitive.

Measuring the quantity of antigen (such as A1AG or A1AT) may be achievedby a variety of methods. One of the most common is to label either theantigen or antibody with a detectable label. Specific, non-limitingexamples of labels include fluorescent tags, enzymatic linkages, andradioactive isotopes (for example ¹⁴C, ³²P, ¹²⁵I, and ³H isotopes andthe like). In some examples, an antibody that specifically binds one ofan antigen of interest is labeled. Methods for labeling and guidance inthe choice of labels appropriate for various purposes are discussed forexample in Sambrook et al. (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., 1989) Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), and Harlow &Lane, (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, 1988)).

Isolated: An isolated biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, for example theseparation of a peptide from a sample, such as saliva, urine, serum orblood. Peptides and proteins that have been isolated include proteinspurified by standard purification methods, such as chromatography, forexample high performance liquid chromatography (HPLC) and the like. Theterm also embraces peptides, and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized peptide andnucleic acids. It is understood that the term “isolated” does not implythat the biological component is free of trace contamination, and mayinclude molecules that are at least 50% isolated, such as at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100%isolated.

Label: Any molecule or composition bound to an analyte, analyte analog,detector reagent, or binding partner that is detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Examples of labels, including enzymes,colloidal gold particles, colored latex particles, have been disclosed(U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and 4,954,452, eachincorporated by reference herein). Additional examples of useful labelsinclude, without limitation, radioactive isotopes, co-factors, ligands,chemiluminescent or fluorescent agents, protein-adsorbed silverparticles, protein-adsorbed iron particles, protein-adsorbed copperparticles, protein-adsorbed selenium particles, protein-adsorbed sulfurparticles, protein-adsorbed tellurium particles, protein-adsorbed carbonparticles, and protein-coupled dye sacs. The attachment of a compound(e.g., a detector reagent) to a label may be through covalent bonds,adsorption processes, hydrophobic and/or electrostatic bonds, as inchelates and the like, or combinations of these bonds and interactionsand/or may involve a linking group.

In some examples, a label is conjugated directly or indirectly toanother molecule, such as an antibody or a protein, to facilitatedetection of that molecule. Specific, non-limiting examples of labelsinclude fluorescent tags, enzymatic linkages, and radioactive isotopes(for example ¹⁴C, ³²P, ¹²⁵I, ³H isotopes and the like). In some examplesa lectin is labeled with a detectable marker, such as biotin. In someexamples an antibody that specifically binds the lectin is labeled witha detectable marker, such as horseradish peroxidase. Methods forlabeling and guidance in the choice of labels appropriate for variouspurposes are discussed for example in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998), Harlow & Lane (Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York, 1988).

Lateral flow device: A device that absorbs or adsorbs a liquid sample,routes that liquid sample to a detection zone, and uses antibody- orlectin-based detection methods to generate a visible signal in responseto the presence or absence of a specific antigen (such as a protein orglycoprotein) or lectin-binding biomolecule (such as a glycoprotein orglycolipid). The device may be a test strip used in lateral flowchromatography, in which a test sample fluid, suspected of containing ananalyte, flows (for example by capillary action) through the strip(which is frequently made of bibulous materials such as paper,nitrocellulose, and cellulose). The test fluid and any suspended analytemay flow along the strip to a detection zone in which the analyte (ifpresent) interacts with a detection agent to indicate a presence,absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,258,548; 6,555,390;6,699,722; and 6,368,876; EP 0810436; and WO 92/12428; WO 94/01775; WO95/16207; and WO 97/06439, each of which is incorporated by reference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials may be used, and rendered bibulous by applying asurfactant to the material), and allowed to migrate along the stripuntil the liquid comes into contact with a specific binding partner(such as a lectin or antibody) that interacts with an analyte (such as aglycoprotein, glycolipid, or antigen) in the liquid. Once the analyteinteracts with the binding partner, a signal (such as a fluorescent orotherwise visible dye) indicates that the interaction has occurred.Multiple discrete binding partners may be placed on the strip (forexample in parallel lines) to detect multiple analytes in the liquid.The test strips may also incorporate control indicators, which provide asignal that the test has adequately been performed, even if a positivesignal indicating the presence (or absence) of an analyte is not seen onthe strip.

Lectin: A carbohydrate-binding protein, some of which are specific forone or more particular carbohydrate moieties. Most known lectins aremultimeric, consisting of non-covalently associated subunits. A lectinmay contain two or more of the same subunit, such as Concanavalin A(ConA), or different subunits, such as Phaseolus vulgaris agglutinin(PHA-E). In particular examples, lectins include Aleuria aurantia lectin(AAL), Concanavalin A (Con A), Datura stramonium lectin (DSL), Erythrinacristagalli lectin (ECL), Griffonia simplicifolia lectin II (GSL-2),Hippeastrum hybrid lectin (HHL), Lycopersicon esculentum lectin (LEL),Lotus tetragonolobus lectin (LTL), Maackia amurensis lectin I (MAL),Phaseolus vulgaris Agglutinin (PHA-E), Sambucus nigra lectin (SNA), andVicia villosa lectin (VVL).

Linking group: A chemical arm between two compounds, for instance acompound and a label (e.g., an analyte, such as a glycoprotein or aglycolipid, and a label). To accomplish the requisite chemicalstructure, each of the reactants must contain a reactive group.Representative combinations of such groups are amino with carboxyl toform amide linkages; carboxy with hydroxy to form ester linkages oramino with alkyl halides to form alkylamino linkages; thiols with thiolsto form disulfides; or thiols with maleimides or alkylhalides to formthioethers. Hydroxyl, carboxyl, amino and other functionalities, wherenot present in the native compound, may be introduced by known methods.

Likewise, a wide variety of linking groups may be employed. Thestructure of the linkage should be a stable covalent linkage formed toattach two compounds to each other (e.g., the label to the analyte). Insome cases the linking group may be designed to be either hydrophilic orhydrophobic in order to enhance the desired binding characteristics, forinstance of the modified ligand and its cognate receptor. The covalentlinkages should be stable relative to the solution conditions to whichlinked compounds are subjected. Examples of linking groups will be from1-20 carbons and 0-10 heteroatoms (NH, O, S) and may be branched orstraight chain. Without limiting the foregoing, it should be obviousthat only combinations of atoms that are chemically compatible comprisethe linking group. For example, amide, ester, thioether, thiol ester,keto, hydroxyl, carboxyl, ether groups in combinations withcarbon-carbon bonds are particular examples of chemically compatiblelinking groups.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomermay be used, the L-isomers being preferred. The terms “polypeptide” or“protein” or “peptide” as used herein are intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” or “protein” or “peptide” isspecifically intended to cover naturally occurring proteins, as well asthose which are produced by recombinant or synthetic methods.

Operable or contiguous contact: Two solid components are in operablecontact when they are in contact, either directly or indirectly, in sucha manner that an aqueous liquid may flow from one of the two componentsto the other substantially uninterruptedly, by capillarity or otherwise.Direct or contiguous contact means that the two elements are in physicalcontact, such as edge-to-edge or front-to-back. When two components arein direct contact, they may overlap with an overlap of about 0.5 mm toabout 3 mm. However, the components may be placed with abutting edges.“Indirect contact” means that the two elements are not in physicalcontact, but are bridged by one or more conductors. Operable contact mayalso be referred to as “fluid transmitting” or “fluid continuous”contact.

Pre-diabetes: A condition identified in a subject by impaired glucosetolerance, alone or in combination with impaired fasting glucoseregulation. An oral glucose tolerance test (OGTT) may be used todetermine if a subject has impaired glucose tolerance (IGT). An OGTTtwo-hour plasma glucose of greater than or equal to 140 mg/dL and lessthan 200 mg/dL (7.8-11.0 mM) is considered to be IGT, and indicates thata subject has pre-diabetes. An OGTT of greater than or equal to 200mg/dl indicates that a subject has frank diabetes, and an OGTT of lessthan 140 mg/dl indicates that a subject is normal (healthy) and does nothave pre-diabetes or diabetes.

Generally, impaired fasting glucose (IFG) may also be used to identify asubject as pre-diabetic. Fasting plasma glucose (FPG) of greater than100 mg/dL and less than 126 mg/dL (5.6-6.9 mM) indicates that a subjecthas IFG and has pre-diabetes. FPG of greater than or equal to 126 mg/dlindicates that a subject has frank diabetes, and an FPG of equal to orless than 100 mg/dl indices that subject is normal (healthy) and doesnot have pre-diabetes or diabetes.

Sample application area: An area where a fluid sample is introduced to aimmunochromatographic test strip, such as an immunochromatographic teststrip present in a lateral flow device. In one example, the sample maybe introduced to the sample application area by external application, aswith a dropper or other applicator. In another example, the sampleapplication area may be directly immersed in the sample, such as when atest strip is dipped into a container holding a sample. In yet anotherexample, the sample may be poured or expressed onto the sampleapplication area.

Solid support (or substrate): Any material which is insoluble, or may bemade insoluble by a subsequent reaction. Numerous and varied solidsupports are known to those in the art and include, without limitation,nitrocellulose, the walls of wells of a reaction tray, multi-wellplates, test tubes, polystyrene beads, magnetic beads, membranes, andmicroparticles (such as latex particles). Any suitable porous materialwith sufficient porosity to allow access by detector reagents and asuitable surface affinity to immobilize capture reagents (e.g., lectinsor antibodies) is contemplated by this term. For example, the porousstructure of nitrocellulose has excellent absorption and adsorptionqualities for a wide variety of reagents, for instance, capturereagents. Nylon possesses similar characteristics and is also suitable.Microporous structures are useful, as are materials with gel structurein the hydrated state.

Further examples of useful solid supports include: natural polymericcarbohydrates and their synthetically modified, cross-linked orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers which may be prepared with suitably porousstructures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

It is contemplated that porous solid supports, such as nitrocellulose,described herein are preferably in the form of sheets or strips. Thethickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, may also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

The surface of a solid support may be activated by chemical processesthat cause covalent linkage of an agent (e.g., a capture reagent) to thesupport. However, any other suitable method may be used for immobilizingan agent (e.g., a capture reagent) to a solid support including, withoutlimitation, ionic interactions, hydrophobic interactions, covalentinteractions and the like. The particular forces that result inimmobilization of an agent on a solid phase are not important for themethods and devices described herein.

A solid phase may be chosen for its intrinsic ability to attract andimmobilize an agent, such as a capture reagent. Alternatively, the solidphase may possess a factor that has the ability to attract andimmobilize an agent, such as a capture reagent. The factor may include acharged substance that is oppositely charged with respect to, forexample, the capture reagent itself or to a charged substance conjugatedto the capture reagent. In another embodiment, a specific binding membermay be immobilized upon the solid phase to immobilize its bindingpartner (e.g., a capture reagent). In this example, therefore, thespecific binding member enables the indirect binding of the capturereagent to a solid phase material.

Except as otherwise physically constrained, a solid support may be usedin any suitable shapes, such as films, sheets, strips, or plates, or itmay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

A “lateral flow substrate” is any solid support or substrate that isuseful in a lateral flow device.

Specific binding partner (or binding partner): A member of a pair ofmolecules that interact by means of specific, noncovalent interactionsthat depend on the three-dimensional structures of the moleculesinvolved. Typical pairs of specific binding partners includeantigen/antibody, carbohydrate/lectin, hapten/antibody,hormone/receptor, nucleic acid strand/complementary nucleic acid strand,substrate/enzyme, inhibitor/enzyme, biotin/(strept)avidin, andvirus/cellular receptor.

The phrase “specifically binds to an analyte” or “specificallyimmunoreactive with,” refers to a binding reaction which isdeterminative of the presence of the analyte in the presence of aheterogeneous population of molecules such as proteins and otherbiologic molecules. Thus, under designated assay conditions, the lectinsor antibodies bind to a particular analyte, such as proteins,glycoproteins and/or glycolipids, and do not bind in a significantamount to other analytes present in the sample. A variety of assayformats may be used to select lectins or antibodies specificallyreactive with a particular analyte, such as a glycoprotein or glycolipidor a particular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow and Lane, Antibodies, ALaboratory Manual, CSHP, New York (1988), for a description ofimmunoassay formats and conditions that may be used to determinespecific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals (such as laboratory or veterinarysubjects).

Substantially the same: An amount that is not significantly differentfrom a reference or control. This may be measure quantitatively, such asby using statistical methods, for example a Student's T-test or ANOVA.For example, a glycosylation profile of a sample from a subject issubstantially the same as a glycosylation profile of a sample from acontrol subject when the amount of glycosylation detected by one or morelectins is not different between the two samples.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein may be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. AllGenBank Accession Nos. mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including explanations of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

III. Methods for Identifying a Subject with Pre-Diabetes or Diabetes

In various embodiments, methods are disclosed herein that may be of useto determine whether a subject has a diabetic condition, for instancepre-diabetes or diabetes. In some embodiments, these methods may utilizea biological sample (such as urine, saliva, blood, serum, amnioticfluid, or tears, for example, saliva, urine, or serum), for thedetection of a glycosylation profile of a plurality of biomolecules(such as glycoproteins or glycolipids) in the sample. In otherembodiments, the glycosylation pattern of one or more specific proteins(such as A1AG or A1AT) may also be used to determine if a subject has adiabetic condition, such as pre-diabetes or diabetes. In some examples,the methods also may include utilizing a biological sample (for exampleurine or saliva) for determining the amount of one or more proteins,including, but not limited to, A1AG or A1AT. In additional examples, themethods also may include determining the glycosylation pattern of A1AGor A1AT.

A. Glycosylation Profile

In some embodiments, the methods disclosed herein may includedetermining a glycosylation profile (such as the amount or type ofglycosylation of a plurality of biomolecules) of a biological sample(such as a biological fluid, for example, urine or saliva). Inembodiments, a change in the glycosylation profile of a sample mayreflect a change in the amount or type of glycosylation (such asN-glycosylation) of a particular biomolecule(s) as compared to theamount or type of the particular biomolecule(s) in a reference standard,such as a reference sample or quantity. A change in the glycosylationprofile may also reflect the presence of glycosylation (such asN-glycosylation) of a particular biomolecule(s) that is not present onthe particular biomolecule(s) in a reference, or the absence ofglycosylation (such as N-glycosylation) of a particular biomolecule(s)that is present on the particular biomolecule(s) in a reference. Thus,in various embodiments, the glycosylation profile may not provideinformation about modification of any one specific biomolecule in thesample, but may provide information about the amount or type ofglycosylation (such as N-glycosylation) present on any biomoleculepresent in the sample or on a population of biomolecules in the sample.

In some embodiments, the glycosylation profile of a sample is determinedby measuring the binding of biomolecules in a biological sample to oneor more lectins. In various embodiments, lectins may bind specificallyto particular carbohydrates or oligosaccharide structures, such as thosepresent in glycoproteins or glycolipids (see, e.g., Section V). In someexamples, a biological sample (such as urine, saliva, or serum) whichcontains a plurality of biomolecules may be contacted with one or morelectins, and binding of biomolecule(s) to the lectin(s) is detected. Invarious embodiments, the amount of biomolecule(s) binding to a lectinhaving a particular specificity indicates the amount of that particulartype of glycosylation present on the plurality of biomolecule(s) in thesample.

In some embodiments, the method includes comparing a glycosylationprofile of a test sample (such as urine, saliva, or serum) from asubject of interest with a glycosylation profile of a referencestandard, such as a reference sample or quantity. In one embodiment, themethod may determine whether the subject has pre-diabetes or diabetes.In embodiments, if the reference is a normal reference and theglycosylation profile of the test sample is substantially the same asthe glycosylation profile of the reference (for example, the amount ortype of glycosylation is substantially the same), the subject isdetermined not to have pre-diabetes or diabetes, respectively. However,in embodiments, if the glycosylation profile of the test sample ischanged relative to the glycosylation profile of the reference (forexample, the amount or type of glycosylation is increased), the subjectis determined to have pre-diabetes or diabetes, respectively.

In another embodiment, if the reference is a pre-diabetes or diabetesreference and the glycosylation profile of the test sample issubstantially the same as the reference sample (for example the amountor type of glycosylation is essentially the same, such as notsignificantly different), then the subject may be determined to havepre-diabetes or diabetes, respectively. In embodiments, of theglycosylation profile of the test sample is changed (for example, adecrease in the amount or type of glycosylation) relative to thereference, the subject is determined not to have pre-diabetes ordiabetes, respectively.

In particular examples, the method may include detecting an increase,such as at least about a 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20-foldincrease (for example about a 2-fold to 20-fold, 3-fold to 15-fold, or5-fold to 10-fold increase), in at least one informative parameter ofthe glycosylation profile, such as the amount of one or more type ofglycosylation (such as N-glycosylation, for example, one or morecarbohydrate group, such as N-acetylglucosamine, N-acetylgalactosamine,N-acetyllactosamine, N-acetylneuraminic acid, mannose, fucose, orgalactose), or the amount of one or more oligosaccharide structures(such as a bi-antennary, tri-antennary, or tetra-antennary structure)present in a sample. An “informative parameter” refers to a measure(such as a quantity of biomolecule binding to a particular lectin) thatdetects a type of glycosylation associated with pre-diabetes ordiabetes.

In particular examples, the amount of glycosylation may be detected bydetermining an amount of a sample binding to a lectin (such as PHA-E,LEL, DSL, ConA, AAL, SNA, or MAL). In some examples, the method mayinclude detecting an increase in biomolecule glycosylation recognized bythe lectin PHA-E (such as at least about a 2, 3, 4, or 5-fold increase,for example about a 2-fold or 3-fold increase), such as lactosamineglycosylation (for example, Gal-β1,4 GlcNAc), in a subject withpre-diabetes or diabetes. In additional examples, the method may includedetecting an increase in biomolecule glycosylation recognized by thelectin LEL (such as at least about a 2, 3, 4, or 5-fold increase, forexample about a 2-fold or 3-fold increase), such as trimers or tetramersof GlcNAc, in a subject with pre-diabetes or diabetes. In additionalexamples, the method may include detecting an increase in biomoleculeglycosylation recognized by the lectin AAL (such as at least about a 2,3, 4, or 5-fold increase, for example about a 2-fold or 3-foldincrease), such as fucose, in a subject with pre-diabetes or diabetes.

In further examples, the method may include detecting an increase inbiomolecule glycosylation recognized by the lectin DSL (such as at leastabout a 2, 3, 4, or 5-fold increase, for example about a 2-fold or3-fold increase) in a subject with pre-diabetes or diabetes. Inadditional examples, the method may include detecting an increase inbiomolecule glycosylation recognized by the lectin ConA (such as atleast about a 2-fold to 20-fold increase, for example about a 2-fold,5-fold, 10-fold, 15-fold, or 20-fold increase) in a subject withpre-diabetes or diabetes. In still further examples, the method mayinclude detecting an increase in biomolecule glycosylation recognized bythe lectin SNA (such as at least about a 2-fold to 15-fold increase, forexample about a 2-fold, 5-fold, 10-fold, or 15-fold increase) in asubject with pre-diabetes or diabetes. In some examples, a glycosylationprofile may include the amount of sample binding to PHA-E, LEL, AAL,DSL, ConA, SNA, MAL, or a combination of two or more thereof.

In still further examples, the amount of the increase of glycosylationdetected by sample binding to a lectin (such as ConA or SNA) may beuseful to differentiate whether a subject has pre-diabetes or diabetes.For example, an increase in sample binding to ConA of about 2-fold to8-fold (such as about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or8-fold) may indicate that the subject has pre-diabetes, while anincrease in sample binding to ConA of about 10-fold to 20-fold (such asabout 10-fold, 12-fold, 15-fold, 18-fold, or 20-fold) may indicate thatthe subject has diabetes. In another example, an increase in samplebinding to SNA of about 2-fold to about 5-fold (such as about 2-fold,3-fold, 4-fold, or 5-fold) may indicate that the subject haspre-diabetes, while an increase in sample binding to SNA of about 8-foldto about 15-fold (such as about 8-fold, 9-fold, 10-fold, 12-fold, or15-fold) may indicate that the subject has diabetes.

In an additional embodiment, the method may include detecting anincrease in a ratio of sample binding to two lectins, such as a testlectin and a reference lectin. In some examples the test lectin mayinclude SNA or ConA, while the reference lectin may include MAL or ECL.In particular examples, an increase in the ratio of SNA binding to MALbinding by a sample (such as an increase of about 2-fold to about20-fold) compared to a normal reference standard, such as a referencesample or quantity, may indicate that the subject has pre-diabetes ordiabetes. In additional examples, an increase in the ratio of SNAbinding to MAL binding of about 2-fold to 5-fold (for example, about2-fold, 3-fold, 4-fold, or 5-fold) compared to a normal referencestandard may indicate that the subject has pre-diabetes. In otherexamples, an increase in the ratio of SNA binding to MAL binding ofabout 10-fold to 15-fold (for example, about 10-fold, 11-fold, 12-fold,13-fold, 14-fold, or 15-fold) compared to a normal reference standardmay indicate that the subject has diabetes.

In further examples, an increase in the ratio of ConA binding to MALbinding by a sample (such as an increase of about 2-fold to about20-fold) compared to a normal reference standard, such as a referencesample or quantity, may indicate that the subject has pre-diabetes ordiabetes. In some examples, an increase in the ratio of ConA binding toMAL binding of about 3-fold to 8-fold (for example, about 3-fold,4-fold, 5-fold, 6-fold, 7-fold, or 8-fold) compared to a normalreference standard may indicate that the subject has pre-diabetes. Inother examples, an increase in the ratio of ConA binding to MAL bindingof about 12-fold to 20-fold (for example, about 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold)compared to a normal reference standard may indicate that the subjecthas diabetes.

Of course, it is not necessary to compare biomolecule glycosylation of asample directly to glycosylation of another sample. Reference standards(such as known quantitative amounts of glycosylation) may be used inlieu of direct comparison to results from a reference sample. Hence, inembodiments, the comparison may be made to a reference (which isunderstood to be results from an actual sample or population of samples,or a qualitative or quantitative reference that represents a cut-offvalue, such as a diagnostic or prognostic level of glycosylation).

B. Amount and Glycosylation of Specific Proteins

In some embodiments, the methods disclosed herein also may includedetermining an amount of one or more proteins present in a biologicalsample (such as urine or saliva), such as proteins associated withpre-diabetes or diabetes. In various embodiments, the methods mayinclude comparing an amount of one or more proteins in a test sample(such as urine or saliva) from a subject of interest with an amount ofsaid protein in a reference standard, such as a reference sample orquantity. In embodiments, if the reference is a normal reference, andthe amount of one or more protein in the test sample is substantiallythe same as the amount of said protein in the reference, the subject maybe determined not to have pre-diabetes or diabetes. However, if theamount of one or more protein of the test sample is changed relative tothe amount of said protein of the reference, the subject may bedetermined to have pre-diabetes or diabetes.

In another embodiment, if the reference is a pre-diabetes or diabetesreference, and the amount of one or more protein in the test sample issubstantially the same as the amount of said protein in the reference,then the subject may be determined to have pre-diabetes or diabetes,respectively. If the amount of one or more protein in the test sample ischanged relative to the reference, the subject may be determined not tohave pre-diabetes or diabetes, respectively. Hence, the amount of one ormore protein may provide an additional diagnostic criterion for thesedisorders.

In some examples, the method may include detecting an increase, such asat least a 2-fold to 10-fold increase (such as at least about a 2, 3, 4,5, 6, 8, or 10-fold increase) in the amount of one or more proteinspresent in a biological sample. In particular examples, the method mayinclude detecting an increase in the amount of A1AG and/or A1AT proteinin a sample from a subject with pre-diabetes or diabetes, as compared toa control sample. In additional examples, the method may includedetecting an increase or decrease, such as at least a 2, 3, 4, or 5-foldincrease or decrease, in the ratio of the amount of A1AG to A1AT in thesample. In particular examples, the ratio of A1AG to A1AT in a samplefrom a subject with diabetes may be decreased (such as about a 2-fold to5-fold decrease, for example about a 2-fold to 3-fold decrease) comparedto a control sample.

In yet another embodiment, the methods disclosed herein also may includedetermining the glycosylation pattern of one or more particular proteins(such as proteins associated with pre-diabetes or diabetes, for exampleA1AG or A1AT) in a biological sample. The glycosylation pattern of aprotein may be determined by detecting binding of the protein to one ormore lectins. In some embodiments, the glycosylation pattern of aprotein may be determined by measuring the binding of the protein in abiological sample to one or more lectins, such as lectins that detect atype of glycosylation of the protein that has been found to beassociated with pre-diabetes or diabetes. The presence (or change, suchas increase or decrease) of one or more particular types ofglycosylation as detected by binding to a lectin specific for type ofglycosylation on the protein may be the glycosylation pattern of theprotein in this example.

In some embodiments, the method may include comparing a glycosylationpattern of one or more proteins in a test sample (such as urine orsaliva) from a subject of interest with a glycosylation pattern of saidproteins from a reference standard, such as a reference sample orquantity. In various embodiments, if the reference is a normal referenceand the glycosylation pattern of one or more protein of the test sampleis substantially the same as the glycosylation pattern of said proteinof the reference, the subject may be determined not to have pre-diabetesor diabetes. However, if the glycosylation pattern of one or moreprotein of the test sample is changed relative to the glycosylationpattern of said protein of the reference, the subject may be determinedto have pre-diabetes or diabetes.

In another embodiment, if the reference is a pre-diabetes or diabetesreference, and the glycosylation pattern of one or more proteins in thetest sample is substantially the same as the glycosylation pattern ofsaid proteins in the reference, then the subject may be determined tohave pre-diabetes or diabetes, respectively. In embodiments, if theglycosylation pattern of one or more protein in the test sample ischanged relative to the reference, the subject may be determined not tohave pre-diabetes or diabetes, respectively.

In some examples, the method may include detecting an increase, such asat least a 2, 3, 4, or 5 fold increase, in the amount or type ofglycosylation of one or more proteins present in the sample. In aparticular example, the method may include detecting an increase (suchas at least about a 2, 3, 4, or 5-fold increase, for example about a2-fold or 3-fold increase) in the amount of glycosylated A1AG recognizedby the lectin ConA in the sample in a subject with pre-diabetes. Inanother particular example, the method may include detecting an increasein the amount of glycosylated A1AG recognized by the lectin SNA (such asat least about a 2, 3, 4, or 5-fold increase, for example about a 2-foldor 3-fold increase) in the sample in a subject with pre-diabetes. In anadditional example, the method may include detecting an increase in theamount of glycosylated A1AG recognized by the lectin AAL (such as atleast about a 2, 3, 4, or 5-fold increase, for example about a 2-fold or3-fold increase) in the sample in a subject with diabetes. Thus, in someexamples, the glycosylation pattern of A1AG may include the amount ofA1AG binding to particular lectins (such as ConA, SNA, AAL, or acombination of two or more thereof).

Of course, it may not be not necessary to compare the amount of one ormore proteins associated with pre-diabetes or diabetes of a sampledirectly to the amount of the proteins of another sample. Likewise, itmay not be necessary to compare the glycosylation pattern of a proteinassociated with pre-diabetes or diabetes directly to the glycosylationpattern of the protein of another sample. Reference standards (such asknown quantitative amounts of glycosylation or a particular protein) maybe used in various embodiments in lieu of direct comparison to resultsfrom a reference sample. Hence, the comparison may be made to a normalreference (which may be understood to be results from an actual sampleor population of samples, or a qualitative or quantitative referencethat represents a cut-off value, such as a diagnostic or prognosticlevel of glycosylation or a particular protein).

In additional embodiments, the glycosylation profile of a sample from asubject may be determined, and the amount and/or glycosylation patternof one or more particular proteins may be determined (such assequentially or simultaneously). In one example, the glycosylationprofile and the amount of one or more proteins may be determined in asample from a subject. In another example, the glycosylation profile andthe glycosylation pattern of one or more proteins may be determined in asample from a subject. In a further example, the glycosylation profile,and the amount and glycosylation pattern of one or more proteins may bedetermined in a sample from a subject.

IV. Methods for Identifying a Subject with Diabetic Complications

In another embodiment, the method may be a method to determine if asubject has a diabetic complication. In particular examples, thediabetic complication may be a chronic complication. Chronic elevationof blood glucose level leads to damage of blood vessels (angiopathy) andchronic diabetic complications. The resulting chronic complications aregrouped as “microvascular complications” (due to damage to small bloodvessels) and “macrovascular complications” (due to damage to thearteries).

Microvascular diabetic complications include (but are not limited to)diabetic nephropathy (such as damage to the kidney which may lead tochronic renal failure), diabetic retinopathy (such as growth of friable,poor-quality blood vessels in the retina which may lead to vision loss),diabetic neuropathy (such as abnormal and decreased sensation, usuallyin a “glove and stocking” distribution), and diabetic cardiomyopathy(damage to the heart leading to diastolic dysfunction and eventuallyheart failure).

Macrovascular complications may lead to cardiovascular disease, to whichaccelerated atherosclerosis may be a contributor. Macrovascularcomplications include (but are not limited to) coronary artery disease(such as angina or myocardial infarction), stroke, peripheral vasculardisease, and diabetic myonecrosis (which may lead to muscle wasting).

Additional diabetic complications may include “diabetic foot” which maylead to skin ulcers, infection, necrosis, and/or gangrene. Thiscomplication may be due to a combination of diabetic neuropathy (such asnumbness or insensitivity) and vascular damage. Diabetic encephalopathy(such as cognitive decline or dementia) is another diabeticcomplication.

The methods described herein include methods to determine whether asubject has one or more diabetic complications. In some embodiments, themethod may include detecting an increase (such as at least about a5-fold increase to about 100-fold increase, for example, about a 5-fold,10-fold, 25-fold, 50-fold, 60-fold, or 100-fold increase) in theglycosylation profile, such as the amount of one or more types ofglycosylation (for example, one or more carbohydrate groups, such asN-acetylglucosamine, N-acetylgalactosamine, N-acetyllactosamine,N-acetylneuraminic acid, mannose, fucose, or galactose) or an amount ofone or more oligosaccharide structures (such as a bi-antennary,tri-antennary, or tetra-antennary structure) present in a sample. Insome embodiments, the method may include comparing a glycosylationprofile of a test sample (such as urine or saliva) from a subject ofinterest with a glycosylation profile of a reference standard, such as areference sample or quantity. In some examples, the method may includedetecting an increase in glycosylation recognized by the lectin AAL(such as at least about 20-fold to 100-fold increase, for example, abouta 25-fold, 50-fold, 60-fold, or 100-fold increase), such as mannose, ina subject with a diabetic complication. In additional examples, themethod may include detecting an increase in glycosylation recognized bythe lectin SNA (such as at least about a 20-fold to 100-fold increase,for example, about a 25-fold, 50-fold, 60-fold, or 100-fold increase),such as N-acetylneuraminic acid, in a subject with a diabeticcomplication.

In one embodiment, if the reference is a normal reference, and theglycosylation profile of the test sample is substantially the same asthe glycosylation profile of the normal sample or reference (forexample, the amount or type of glycosylation is substantially the same),the subject may be determined not to have a diabetic complication.However, if the glycosylation profile of the test sample is changedrelative to the glycosylation profile of the normal sample or reference(for example, the amount of one or more type of glycosylation isincreased), the subject may be determined to have a diabeticcomplication. In a particular example, the diabetic complication may bea microvascular complication, for example, diabetic nephropathy,diabetic retinopathy, or diabetic neuropathy.

In another embodiment, if the reference is a diabetic complicationreference, and the glycosylation profile of the test sample issubstantially the same as the reference (for example the amount or typeof glycosylation is essentially the same, such as not significantlydifferent), then the subject may be determined to have a diabeticcomplication. If the glycosylation profile of the test sample is changed(for example, a decrease in the amount or type of glycosylation)relative to the reference, then the subject may be determined not tohave a diabetic complication.

In additional examples, the method for identifying a subject as having adiabetic complication may include determining the amount of one or moreproteins (such as proteins associated with diabetic complication, suchas A1AG or A1AT) in a sample from the subject. In embodiments, themethods may include comparing an amount of one or more proteins in atest sample (such as urine or saliva) from a subject of interest with anamount of said protein from a reference standard, such as a referencesample or quantity. In particular examples, the method may includedetecting an increase in protein amount (such as at least about a20-fold increase to about 1000-fold increase, such as about a 20-fold,50-fold, 70-fold, 100-fold, 300-fold, 500-fold, or 1000-fold increase)in a subject with a diabetic complication compared to a referencestandard, such as a reference sample or quantity. In additionalexamples, the method may include detecting an increase or decrease, suchas at least a 2, 3, 4, or 5-fold increase or decrease, in the ratio ofthe amount of A1AG to A1AT in the sample. In particular examples, theratio of A1AG to A1AT in a sample from a subject with a diabeticcomplication may be decreased (such as about a 2-fold or 3-folddecrease) compared to a reference standard, such as a reference sampleor quantity.

In various embodiments, if the reference is a normal reference, and theamount of one or more proteins of the test sample is substantially thesame as the amount of said protein in the normal sample, the subject maybe determined not to have a diabetic complication. However, if theamount of one or more proteins of the test sample is changed relative tothe amount of said protein of the normal sample or standard, the subjectis determined to have a diabetic complication.

In another embodiment, if the reference is a diabetic complicationreference, and the amount of one or more protein in the test sample issubstantially the same as the amount of said protein in the reference,then the subject may be determined to have a diabetic complication. Ifthe amount of one or more proteins in the test sample is changedrelative to the reference, the subject is determined not to have adiabetic complication.

In additional embodiments, the glycosylation profile of a sample from asubject may be determined, and the amount and/or glycosylation patternof one or more particular proteins may be determined (such assequentially or simultaneously). In one example, the glycosylationprofile and the amount of one or more proteins may be determined in asample from a subject. In another example, the glycosylation profile andthe glycosylation pattern of one or more proteins may be determined in asample from a subject. In a further example, the glycosylation profile,and the amount and glycosylation pattern of one or more proteins may bedetermined in a sample from a subject.

V. Monitoring

In another embodiment, the method may be a method to determine whether atherapy is effective for the treatment of the subject. Thus, in variousembodiments, the method may be performed multiple times over a specifiedtime period, such as days, weeks, months or years. The diagnosticmethods described herein are valuable tools for practicing physicians tomake quick treatment decisions for diabetic conditions, includingpre-diabetes and diabetes. These treatment decisions may include thedecision to administration of an anti-diabetic agent, and/or thedecision to monitor a subject for the onset and/or advancement ofdiabetes. The treatment decisions may also include lifestyle monitoring.The method disclosed herein may also be used to monitor theeffectiveness of a therapy.

In various embodiments, the glycosylation profile, protein expressionlevel, or glycosylation pattern of a sample from a subject may beassessed as described above and compared to a reference. If thereference, such as a reference sample or quantity, is a normalreference, and the glycosylation profile (or amount or glycosylationpattern of a particular protein) of the test sample is substantially thesame as the glycosylation profile (or amount or glycosylation pattern ofa particular protein) of the normal reference, the subject may bedetermined to have an effective therapy Conversely, if the glycosylationprofile (or amount or glycosylation pattern of a particular protein) ofthe test sample is different from the glycosylation profile (or amountor glycosylation pattern of a particular protein) of the normalreference, the subject may be determined to have an ineffective therapy.In embodiments, if the reference standard, such as a reference sample orquantity, is a pre-diabetes or diabetes reference, and the glycosylationprofile (or amount or glycosylation pattern of a particular protein)does not differ from the reference, then the subject may be determinedto have an ineffective therapy. Conversely, if the glycosylation profile(or amount or glycosylation pattern of a particular protein) of the testsample is different from the reference, the subject may be determined tohave an effective therapy.

Following the assessment of the glycosylation profile, proteinexpression level, or glycosylation pattern identified herein, the assayresults, findings, diagnoses, predictions and/or treatmentrecommendations may be recorded and/or communicated to technicians,physicians and/or patients, for example. In certain embodiments,computers may be used to communicate such information to interestedparties, such as patients and/or the attending physicians. In variousembodiments, based on the measurement, the therapy administered to asubject may be modified.

In one embodiment, a diagnosis, prediction and/or treatmentrecommendation based on the glycosylation profile, protein expressionlevel, or glycosylation pattern in a test subject of one or more of theglycosylated biomolecules described herein may be communicated to thesubject as soon as possible after the assay is completed and thediagnosis and/or prediction is generated. In some embodiments, theresults and/or related information may be communicated to the subject bythe subject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, such as by providing a written report, electronicforms of communication, such as email, or telephone. In embodiments,communication may be facilitated by use of a computer, such as in caseof email communications. In certain embodiments, the communicationcontaining results of a diagnostic test and/or conclusions drawn fromand/or treatment recommendations based on the test, may be generated anddelivered automatically to the subject using a combination of computerhardware and software which will be familiar to artisans skilled intelecommunications. One example of a healthcare-oriented communicationssystem is described in U.S. Pat. No. 6,283,761; however, the presentmethods are not limited to methods which utilize this particularcommunications system. In certain embodiments of the methods, all orsome of the method steps, including the assaying of samples, diagnosingof diseases, and communicating of assay results or diagnoses, may becarried out in diverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as beingpre-diabetic or diabetic may result in the physician treating thesubject, such as by prescribing an anti-hyperglycemic or ananti-diabetic agent to inhibit or delay the onset or progression of typeII diabetes. In additional embodiments, the dose or dosing regimen maybe modified based on the information obtained using the methodsdisclosed herein. In some embodiments, the anti-diabetic agent maycontain a biguanide of the formula:

wherein R₁ and R₂ are independently selected from alkyl, lower alkyl,alkenyl, lower alkenyl, cycloalkyl, aryl, or an arylalkyl of theformula:

wherein X is hydrogen or halogen and n=0, 1 or 2; R₃ and R₄ areindependently selected from hydrogen, alkyl, lower alkyl, alkenyl, loweralkenyl, cycloalkyl, alkoxy, lower alkoxy, alkoxyalkyl; andpharmaceutically acceptable salts thereof. In particular embodiments,the biguanide antidiabetic agent may be metformin. Metformin may bemanufactured by Lyonnaise Industrielle Pharmaceutique SA (Lyons,France), and is also known by its acronym LIPHA SA, and may becommercially distributed in the United States as a hydrochloride salt bythe Bristol-Myers Squibb Company (Princeton, N.J.) as GLUCOPHAGE® XR.Additionally, Bristol-Myers Squibb currently distributes apharmaceutical having a combination of metformin and glyburide asGLUCOVANCE®.

Anti-diabetic agents other than biguanides may also be administered tothe identified subject. For example, in alternative embodiments, theanti-diabetic agent may be a thiazolidinedione, such as troglitazone. Insome examples, the anti-diabetic agent may be an incretin or dipeptidylpeptidase-4 inhibitor, but in various other examples, the anti-diabeticagent may be any agent of interest.

In embodiments, a therapeutically effective amount of an anti-diabeticagent may be administered in a single dose, or in several doses, forexample daily, during a course of treatment. The course of treatment maylast for any length of time, such as a day or several days, a week orseveral weeks, a month or several months, or a year or several years, solong as the therapeutic effect is observed, such as inhibiting the onsetof type II diabetes in a subject diagnosed with pre-diabetes, orinducing a subject diagnosed with type 2 diabetes or pre-diabetes to anormal glucose tolerance. In various embodiments, the subject may bemonitored while undergoing treatment using the methods described hereinin order to assess the efficacy of the treatment protocol. In thismanner, the length of time or the amount give to the subject may bemodified based on the results obtained using the methods disclosedherein.

In various embodiments, the therapeutically effective amount of theanti-diabetic agent may depend on the anti-diabetic agent being used,the characteristics of the subject being treated (such as age, BMI,physiological condition, etc.), the severity and type of the affliction,and the manner of administration of the agent. The therapeuticallyeffective dose may be determined by various methods, includinggenerating an empirical dose-response curve, predicting potency andefficacy by using quantitative structure activity relationships (QSAR)methods or molecular modeling, and other methods used in thepharmaceutical sciences. In certain, non-limiting examples, thetherapeutically effective amount of metformin (or a related biguanideanalog or homolog) may be at least about 1000 mg per day, such as atleast about 1500 mg per day, or even at least about 1700 mg per day. Incertain other, non-limiting examples, the total amount of metformin maybe divided into smaller doses, such as two or three doses per day, forexample 850 mg twice a day (b.i.d.) or 500 mg three times a day(t.i.d.). In alternative, non-limiting examples, the total amount ofmetformin may be about 500 mg or less per day. In embodiments, thesubject may be monitored at different doses of an agent using the assaysdescribed herein in order to determine a therapeutically effectiveamount for the subject of interest.

For administration to animals or humans, purified therapeutically activeagents are generally combined with a pharmaceutically acceptablecarrier. Pharmaceutical preparations may contain only one type ofanti-diabetic agent, or may be composed of a combination of severaltypes of anti-diabetic agents, such as a combination of two or moreanti-diabetic agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers may include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered may contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Anti-diabetic agents may be administered by any means that achieve theirintended purpose. For example, the anti-diabetic agents may beadministered to a subject through systemic administration, such assubcutaneous, intravenous or intraperitoneal administration, bysuppository, or by oral administration. In embodiments, theanti-diabetic agent may be administered alone or in combination withanother anti-diabetic agent. In certain embodiments, the anti-diabeticagent may be administered in the absence of administering any otheranti-diabetic agent.

In embodiments, other measures may be taken to inhibit or delay theonset of type II diabetes in subjects at a heightened risk of developingthe disease. For example, in some embodiments, a subject may beinstructed, trained, or induced to adopt anti-diabetic lifestylemodifications. For example, the subject may be counseled to reducecaloric intake or to exercise. In various embodiments, the methodsdisclosed herein may be used to monitor the effectiveness of thesealternative measures, for instance, to determine if pharmaceuticalintervention is warranted for a subject of interest.

VI. Lectins

Lectins are carbohydrate-binding proteins, some of which are specificfor particular carbohydrate moieties. The term “lectin” was originallycoined to define agglutinins which could discriminate among types of redblood cells, however the term is now used more generally and includescarbohydrate-binding proteins from many sources regardless of theirability to agglutinate cells. Lectins have been found in plants,viruses, microorganisms and animals. Their function in nature isunclear, however lectins share the common property of binding to definedsugar structures.

In embodiments, the glycosylation profile of a sample may be determinedby binding to one or more lectins. In some embodiments, theglycosylation profile may be expressed in terms of the lectins to whicha sample binds (such as the lectins provided in Table 1, below). Theglycosylation profile may also be expressed in terms of the particularcarbohydrate modifications present in the sample, based on thecarbohydrate-binding specificity of the particular lectins (such asdescribed in Table 1, below) to which the sample binds. However, inembodiments, the identification of specific carbohydrates in a samplemay not be essential to determining the glycosylation profile of thesample in the methods described herein.

Similarly, in some embodiments, the glycosylation pattern of aparticular protein may be determined by binding to one or more lectins.In some embodiments, the glycosylation pattern of a protein may beexpressed in terms of the lectins to which the protein binds (such asthe lectins provided in Table 1, below). The glycosylation pattern ofthe protein may also be expressed in terms of the particularcarbohydrate modifications present on the protein, based on thecarbohydrate-binding specificity of the particular lectins (such asdescribed in Table 1, below) to which the protein binds, or by othermethods known in the art (such as mass spectrometry). However, inembodiments, the identification of specific carbohydrates on the proteinmay not be essential to determining the glycosylation pattern of theprotein in the methods described herein.

Lectins are well known to those of skill in the art. In particularexamples, lectins may include Aleuria aurantia lectin (AAL),Concanavalin A (Con A), Datura stramonium lectin (DSL), Erythrinacristagalli lectin (ECL), Griffonia simplicifolia lectin II (GSL-2),Hippeastrum hybrid lectin (HHL), Lycopersicon esculentum lectin (LEL),Lotus tetragonolobus lectin (LTL), Maackia amurensis lectin I (MAL),Phaseolus vulgaris Agglutinin (PHA-E), Sambucus nigra lectin (SNA), andVicia villosa lectin (VVL). The binding specificity of exemplary lectinsis presented in Table 1, however, the table is not exhaustive and thelectins may also bind to additional carbohydrate modifications.

TABLE 1 Exemplary lectins and binding specificity Lectin Bindingspecificity Aleuria aurantia lectin (AAL) Fucose Concanavalin A (Con A)Mannose>glucose Datura stramonium lectin (DSL) Gal β1,4 GlcNAc,oligomers of GlcNAc and LacNAc Erythrina cristagalli lectin (ECL)β-gal(1→4)GlcNAc; galactose and galactosides Griffonia simplicifolialectin II (GSL-2) terminal α-D-galactosyl residues, terminalN-acetyl-α-D-galactosaminyl residues Hippeastrum hybrid lectin (HHL)Mannose Lycopersicon esculentum lectin (LEL) GlcNAc oligomers Lotustetragonolobus lectin (LTL) Fucose Maackia amurensis lectin I (MAL)N-acetylneuraminic acid (sialic acid) Phaseolus vulgaris Agglutinin(PHA- Gal-β1,4 GlcNAc E) Sambucus nigra lectin (SNA) α-NeuNAc-[2→6]-Gal,α- NeuNAc-[2→6]-GalNAc, α-NeuNAc-[2→3]-Gal Vicia villosa lectin (VVL)O-linked GalNAc

Additional lectins are well known to one of skill in the art. In someexamples, lectins may include Agaricus bisporus lectin, Amaranthuscaudatus lectin, Griffonia simplicifolia lectin I, Bauhinia purpureaalba lectin, Codium fragile lectin, Dolichos biflorus lectin, Erythrinacoralldendron lectin, Euonymos europaeus lectin, Glycine max lectin,Helix aspersa lectin, Helix pomatia lectin, Maclura pomifera lectin,Narcissus pseudonarcissus lectin, Phaseolus coccineus lectin, Phaseolusvulgaris L lectin, Phytolacca Americana lectin, Pisum sativum lectin,Psophocarpus tetragonolobus I lectin, Solanum tuberosum lectin, Sophorajaponica terminal lectin, Wisteria floribunda lectin, Anguilla anguillalectin, Arachis hypogaea lectin, Artocarpus integrifolia lectin,Bandeiraea simplicifolia lectin, Caragana arborescens lectin, Cicerarietinum lectin, Galanthus nicalis lectin, Lens culinaris lectin,Limulus polyphemus lectin, Pseudomonas aeruginosa lectin, Ricin communisagglutinin, Triticum vulgaris lectin, Ulex europaeus lectin, Vicia fabalectin, and Visum album lectin. In various embodiments, any lectin thatidentifies a difference between the glycosylation profile of a sample orthe glycosylation pattern of one or more protein from a subject withpre-diabetes, diabetes, or a diabetic complication and a reference maybe used in the disclosed methods.

VII. Detection Methods

The methods disclosed herein may be performed in the form of variousassay formats, which are well known in the art. In some examples, theassays may be antibody-based or lectin-based assays. There are two maintypes of assays, homogenous and heterogenous. In homogenous assays, theimmunological reaction between an antigen and an antibody (or thereaction between an oligosaccharide and a lectin) and the detection arecarried out in a homogenous reaction. Heterogenous assays include atleast one separation step, which allows the differentiation of reactionproducts from unreacted reagents.

ELISA is a heterogenous assay, which has been widely used in laboratorypractice since the early 1970s, and may be used in the methods disclosedherein. The assay may be used to detect protein antigens orcarbohydrates (such as monosaccharides or oligosaccharides linked to aprotein) in various formats. In the “sandwich” format the antigen orcarbohydrate being assayed is held between two different antibodies orlectins, respectively. Another type of sandwich assay holds theglycoprotein between an antibody and a lectin.

A sandwich ELISA may be used to detect the presence or amount of anantigen in a sample, such as a glycoprotein (a “protein ELISA”). In thismethod, a solid surface is first coated with a solid phase antibody. Thetest sample, containing the antigen (such as a diagnostic glycoprotein),or a composition containing the antigen, such as a urine or salivasample from a subject of interest, is then added and the antigen isallowed to react with the bound antibody. Any unbound antigen is washedaway. A known amount of enzyme-labeled antibody is then allowed to reactwith the bound antigen. Any excess unbound enzyme-linked antibody iswashed away after the reaction. The substrate for the enzyme used in theassay is then added and the reaction between the substrate and theenzyme produces a color change. The amount of visual color change is adirect measurement of specific enzyme-conjugated bound antibody, andconsequently the antigen present in the sample tested.

ELISA may also be used to detect the presence or amount carbohydrates(such as monosaccharides or oligosaccharides, for example in the form ofglycoproteins or glycolipids) in a sample by utilizing a lectin in placeof an antibody in the assay. In a “lectin ELISA,” a solid surface iscoated with a lectin. The test sample containing one or moreglycosylated biomolecule, such as a urine or saliva sample from asubject of interest, is then added and the carbohydrate in theglycoprotein is allowed to react with the bound lectin. Any unboundglycosylated biomolecule is washed away and a known amount ofenzyme-labeled lectin is then allowed to react with the bound antigen.Any excess unbound enzyme-linked lectin is washed away after thereaction. The substrate for the enzyme used in the assay is then addedand the reaction between the substrate and the enzyme produces a colorchange. The amount of visual color change is a direct measurement ofspecific enzyme-conjugated bound lectin, and consequently thecarbohydrate present in the sample tested. A lectin ELISA may notidentify the particular biomolecule(s) which is recognized by thelectin, rather a lectin ELISA may provide a glycosylation profile, suchas the amount or type of one or more carbohydrate modifications on oneor more biomolecule that is present in the sample.

In other examples, an “antibody-lectin ELISA” may be used to detect thepresence or amount of carbohydrate modifications on a particular proteinin a sample. In this method, a solid surface is first coated with asolid phase antibody. The test sample, containing the antigen (such asA1AG or A1AT), or a composition containing the antigen, such as a urineor saliva sample from a subject of interest, is then added and theantigen is allowed to react with the bound antibody. Any unbound antigenis washed away. A known amount of enzyme-labeled lectin is then allowedto react with the bound antigen. Any excess unbound enzyme-linked lectinis washed away after the reaction. The substrate for the enzyme used inthe assay is then added and the reaction between the substrate and theenzyme produces a color change. The amount of visual color change is adirect measurement of specific enzyme-conjugated bound lectin, andconsequently the carbohydrate modification present on the particularantigen (the glycosylation pattern of the antigen) in the sample tested.

ELISA may also be used as a competitive assay. In the competitive assayformat, the test specimen containing the antigen or oligosaccharide tobe determined is mixed with a precise amount of enzyme-labeled antigenor oligosaccharide (such as an enzyme-labeled glycoprotein containingthe oligosaccharide) and both compete for binding to an anti-antigenantibody or a lectin attached to a solid surface. Excess freeenzyme-labeled antigen or glycoprotein is washed off before thesubstrate for the enzyme is added. The amount of color intensityresulting from the enzyme-substrate interaction is a measure of theamount of antigen or oligosaccharide in the sample tested.

Similar assays may be performed using alternate platforms for theidentification of the glycosylation profile, amount of particularproteins, or glycosylation pattern of particular proteins. For example,microsphere-based assays may be used to detect binding of an antigen toan antibody or a carbohydrate modification to a lectin. Briefly,microsphere beads are coated with an antibody or a lectin and mixed witha sample, such that an antigen or a carbohydrate modification,respectively, present in the sample that are specifically reactive withthe antibody or lectin bind to the bead. The bead-bound complexes areallowed to react with fluorescent-dye labeled antibody or lectin, andare measured using a microsphere reader (such as a Luminex instrument).Alternatively, the antibody or lectin may be coupled to a magnetic bead,allowing separation and detection of particular proteins or particularcarbohydrate modifications present in the sample.

In additional examples, the glycosylation profile, amount of particularproteins, or glycosylation pattern of particular proteins may bedetermined with methods utilizing antibody or lectin arrays. Forexample, the glycosylation profile of a sample may be determined byapplying the sample to a lectin array including two or more lectins (forexample, two or more of PHA-E, LEL, AAL, ConA, SNA, or MAL) ataddressable locations and detecting binding of the sample to particularlectins. The profile of binding of a sample from a subject to the arraymay be compared to a reference profile (such as a profile from a normalsubject) to determine whether the subject is pre-diabetic, diabetic, orhas a diabetic complication. Similarly, the amount of particularproteins present in a sample may be determined by applying the sample toan antibody array including two or more antibodies (for example A1AG andA1AT) and detecting binding of the sample to particular antibodies. Inadditional examples, the glycosylation pattern of particular proteins(for example, A1AG or A1AT) may be determined by applying a samplecontaining the protein to a lectin array (such as Qproteome GlycoArray,Qiagen, Valencia, Calif.) and detecting binding to particular lectins inthe array with a protein-specific antibody.

Other methods for determining the glycosylation profile, amount ofglucosylated proteins, or glycosylation pattern of particular proteinsmay be used. For example, Western blotting may be used to determine theamount of a particular protein in a sample. Similarly, lectin blottingmay be used to determine the amount of a particular carbohydratemodification present in a sample, thus providing a glycosylation profileof the sample. The glycosylation pattern of a protein may be determined,for example by immunoprecipitation of a particular protein from asample, followed by lectin blotting to determine the carbohydratemodifications present on the protein.

VIII. Capture Device Methods

In various embodiments, the disclosed methods may be carried out using asample capture device, such as a lateral flow device (for example alateral flow test strip) that allows detection of a glycosylationprofile, glycosylation pattern, or amount of protein.

Point-of-use analytical tests have been developed for the routineidentification or monitoring of health-related conditions (such aspregnancy, cancer, endocrine disorders, infectious diseases or drugabuse) using a variety of biological samples (such as urine, serum,plasma, blood, saliva). Some of the point-of-use assays are based onhighly specific interactions between specific binding pairs, such asantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. The assays are oftenperformed with test strips in which a specific binding pair member isattached to a mobilizable material (such as a metal sol or beads made oflatex or glass) or an immobile substrate (such as glass fibers,cellulose strips or nitrocellulose membranes). Particular examples ofsome of these assays are shown in U.S. Pat. Nos. 4,703,017; 4,743,560;and 5,073,484. The test strips may include a flow path from an upstreamsample application area to a test site. For example, the flow path maybe from a sample application area through a mobilization zone to acapture zone. The mobilization zone may contain a mobilizable markerthat interacts with an analyte or analyte analog, and the capture zonemay contain a reagent that binds the analyte or analyte analog to detectthe presence of an analyte in the sample.

Examples of migration assay devices, which usually incorporate withinthem reagents that have been attached to colored labels, therebypermitting visible detection of the assay results without addition offurther substances, are found, for example, in U.S. Pat. No. 4,770,853;WO 88/08534; and EP-A 0 299 428. There are a number of commerciallyavailable lateral-flow type tests and patents disclosing methods for thedetection of large analytes (MW greater than 1,000 Daltons) as theanalyte flows through multiple zones on a test strip. Examples are foundin U.S. Pat. No. 5,229,073 (measuring plasma lipoprotein levels), andU.S. Pat. Nos. 5,591,645; 4,168,146; 4,366,241; 4,855,240; 4,861,711;5,120,643; European Patent No. 0296724; WO 97/06439; and WO 98/36278.Multiple zone lateral flow test strips are disclosed in U.S. Pat. No.5,451,504, U.S. Pat. No. 5,451,507, and U.S. Pat. No. 5,798,273. U.S.Pat. No. 6,656,744 discloses a lateral flow test strip in which a labelbinds to an antibody through a streptavidin-biotin interaction.

In particular examples, the methods disclosed herein may includeapplication of a biological sample (such as saliva or urine) from a testsubject to a lateral flow test device for the detection of aglycosylation profile of the sample. In embodiments, the lateral flowtest device may include one or more lectins (such as one or more ofPHA-E, LEL, AAL, DSL, ConA, SNA, or MAL) at one or more addressablelocations. The addressable locations may be, for example, a linear arrayor other geometric pattern that provides diagnostic information to theuser. The binding of one or more proteins in the sample to the lectinspresent in the test device may be detected, and the glycosylationprofile of the test subject may be compared to the glycosylation profileof a control sample, wherein a change in the glycosylation profile ofthe sample from the test subject as compared to the control sampleindicates that the subject has pre-diabetes, diabetes, or a diabeticcomplication (such as diabetic nephropathy).

In some examples, the methods disclosed herein may further includedetermining the amount of one or more proteins (such as one or moreprotein associated with pre-diabetes or diabetes) in a biological sample(such as saliva or urine) from a test subject by applying the biologicalsample to a lateral flow test device. The test device may include one ormore antibodies (such as anti-A1AG or anti-A1AT) at an addressablelocation In embodiments, the binding of the proteins in the sample fromthe test subject to the particular antibodies may be detected, and theamount of the protein in the sample from the test subject may becompared to the amount of the protein in a control sample, wherein achange in the amount of the protein in the sample from the test subjectas compared to the control sample may indicate that the subject haspre-diabetes, diabetes, or a diabetic complication (such as diabeticnephropathy).

A. Flow-Through Devices

Flow-through type assay devices were designed, in part, to obviate theneed for incubation and washing steps associated with dipstick assays.In embodiments, flow-through immunoassay devices may include a capturereagent (such as one or more lectins) bound to a porous membrane orfilter to which a liquid sample is added. As the liquid flows throughthe membrane, target analyte (such as glycoproteins, glycolipids, orproteins) binds to the capture reagent. The addition of sample isfollowed by (or made concurrent with) addition of detector reagent (suchas, labeled (e.g., gold-conjugated, or colored latexparticle-conjugated) glycoprotein, labeled (e.g., gold-conjugated orcolored latex particle-conjugated) glycolipid or labeled (e.g.,gold-conjugate or colored latex particle-conjugated) protein (such asA1AT or A1AG). Alternatively, the detector reagent may be placed on themembrane in a manner that permits the detector to mix with the sampleand thereby label the analyte. The visual detection of detector reagentprovides an indication of the presence of target analyte in the sample.Representative flow-through assay devices are described in U.S. Pat.Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and5,279,935; and U.S. Patent Application Publication Nos. 20030049857 and20040241876. Migration assay devices usually incorporate within themreagents that have been attached to colored labels, thereby permittingvisible detection of the assay results without addition of furthersubstances. See, for example, U.S. Pat. No. 4,770,853; PCT PublicationNo. WO 88/08534 and European Patent No. EP-A 0 299 428.

There are a number of commercially available lateral flow type tests andpatents disclosing methods for the detection of large analytes (MWgreater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes asemiquantitative competitive immunoassay lateral flow method formeasuring plasma lipoprotein levels. This method utilizes a plurality ofcapture zones or lines containing immobilized antibodies to bind boththe labeled and free lipoprotein to give a semi-quantitative result. Inaddition, U.S. Pat. No. 5,591,645 provides a chromatographic test stripwith at least two portions. The first portion includes a movable tracerand the second portion includes an immobilized binder capable of bindingto the analyte. Additional examples of lateral flow tests for largeanalytes are disclosed in the following patent documents: U.S. Pat. Nos.4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; EuropeanPatent No. 0296724; WO 97/06439; and WO 98/36278. There are also lateralflow type tests for the detection of small analytes (MW 100-1,000Daltons). Generally, these small analyte tests involve “typical”competitive inhibition to produce negative or indirect reporting results(i.e., reduction of signal with increasing analyte concentration), asexemplified by U.S. Pat. No. 4,703,017. However, several approaches havebeen developed for detecting small analytes using lateral flow teststhat produce positive or direct reporting results (i.e., increase insignal with increasing analyte concentration). These include, forinstance, U.S. Pat. Nos. 5,451,504; 5,451,507; 5,798,273; and 6,001,658.U.S. Pat. No. 5,451,504 provides a method with three specific zones(mobilization, trap and detection) each containing a different latexconjugate to yield a positive signal. The mobilization zone containslabeled antibody or lectin to bind the analyte in the sample. In thetrap zone, unbound, labeled antibody or lectin is then trapped byimmobilized analyte analog. The detection zone captures the labeledanalyte-antibody or -lectin complex. U.S. Pat. No. 5,451,507 describes atwo-zone, disconnected assay method. The first zone has non-diffusivelybound reagent that binds with a component, for example, an analyteanalog bound to, or capable of becoming bound to, a member of a signalproducing system. The second zone binds to the component only when theanalyte to be tested is present. The distance the component migratesinto the second zone is directly related to the concentration ofanalyte. U.S. Pat. No. 5,798,273 discloses a lateral flow device thatincludes a capture zone with immobilized analyte analog and one or moreread-out zones to bind labeled analyte-analog. U.S. Pat. No. 6,001,658discloses a test strip device with a diffusible, labeled binding partnerthat binds with analyte, an immobilized analyte, and a detection areacontaining an immobilized antibody.

Devices described herein generally include a strip of absorbent material(such as a microporous membrane), which, in some instances, may be madeof different substances each joined to the other in zones, which may beabutted and/or overlapped. In some examples, the absorbent strip may befixed on a supporting non-interactive material (such as nonwovenpolyester), for example, to provide increased rigidity to the strip.Zones within each strip may differentially contain the specific bindingpartner(s) and/or other reagents required for the detection and/orquantification of the particular analyte being tested for, for example,glycoproteins and/or glycolipids or particular proteins. Thus thesezones may be viewed as functional sectors or functional regions withinthe test device.

In general, a fluid sample is introduced to the strip at the proximalend of the strip, for instance by dipping or spotting. A sample iscollected or obtained using methods well known to those skilled in theart. The sample containing the glycoproteins and/or glycolipids orparticular proteins to be detected may be obtained from any biologicalsource. Examples of biological sources include blood serum, bloodplasma, urine, spinal fluid, saliva, fermentation fluid, lymph fluid,tissue culture fluid and ascites fluid of a human or animal. The samplemay be diluted, purified, concentrated, filtered, dissolved, suspendedor otherwise manipulated prior to assay to optimize the immunoassayresults. The fluid migrates distally through all the functional regionsof the strip. The final distribution of the fluid in the individualfunctional regions depends on the adsorptive capacity and the dimensionsof the materials used.

In some embodiments, porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, may also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

Another common feature to be considered in the use of assay devices is ameans to detect the formation of a complex between an analyte (such as aglycoprotein or a glycolipid or one or more particular proteins) and acapture reagent (such as one or more lectins or antibodies). A detector(also referred to as detector reagent) serves this purpose. A detectormay be integrated into an assay device (for example included in aconjugate pad, as described below), or may be applied to the device froman external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte (such as,gold-conjugated lectin for a glycoprotein analyte or a glycolipidanalyte or a gold-conjugated antibody for a particular protein).Exemplary lectins are listed in Table 1 above (for example, PHA-E, LEL,AAL, DSL, ConA, SNA, and/or MAL). Exemplary antibodies include, but arenot limited to antibodies to A1AG and A1AT. Such antibodies may becommercially available. Exemplary commercially available antibodiesinclude A1AG antibodies (such as catalog numbers sc-59447, sc-51018,sc-51020, Santa Cruz Biotechnology, Santa Cruz, Calif.; catalog numberA0011, Dako, Carpinteria, Calif.; catalog numbers ab440, ab17695, Abcam,Cambridge, Mass.) and A1AT antibodies (such as catalog numbers sc-59435,sc-59436, sc-69986, Santa Cruz Biotechnology, Santa Cruz, Calif.;catalog numbers ab7633, ab9400, ab14226, Abcam, Cambridge, Mass.).

In other instances, a detector reagent collectively includes anunlabeled first binding partner specific for the analyte and a labeledsecond binding partner specific for the first binding partner and soforth. Thus, the detector may be a labeled lectin or antibody specificfor a glycoprotein or a glycolipid or a particular protein. The detectormay also be an unlabeled first lectin or antibody specific for theglycoprotein or glycolipid or a particular protein and a labeled secondantibody that specifically binds the unlabeled first lectin or antibody.In each instance, a detector reagent specifically detects bound analyteof an analyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent may specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

B. Flow-Through Device Construction and Design

A flow-through device may involve a capture reagent (such as one or morelectins) immobilized on a solid support, typically, microtiter plate ora membrane (such as, nitrocellulose, nylon, or PVDF). Characteristics ofuseful membrane have been previously described; however, it is useful tonote that in a flow-through assay, capillary rise is not a particularlyimportant feature of a membrane as the sample moves vertically throughthe membrane rather than across it as in a lateral flow assay. In asimple representative format, the membrane of a flow-through device isplaced in functional or physical contact with an absorbent layer (see,e.g., description of “absorbent pad” below), which acts as a reservoirto draw a fluid sample through the membrane. Optionally, followingimmobilization of a capture reagent, any remaining protein-binding siteson the membrane may be blocked (either before or concurrent with sampleadministration) to minimize nonspecific interactions.

In operation of a flow-through device, a fluid sample (such as a bodilyfluid sample) is placed in contact with the membrane. Typically, aflow-through device also includes a sample application area (orreservoir) to receive and temporarily retain a fluid sample of a desiredvolume. The sample passes through the membrane matrix. In this process,an analyte in the sample (such as a glycoprotein or glycolipid or one ormore particular protein) may specifically bind to the immobilizedcapture reagent (such as one or more lectins or antibodies). Wheredetection of an analyte-capture reagent complex is desired, a detectorreagent (such as labeled lectin or labeled antibodies that specificallybind glycoproteins, glycolipids, or one or more particular protein) maybe added with the sample or a solution containing a detector reagent maybe added subsequent to application of the sample. If an analyte isspecifically bound by capture reagent, a visual representativeattributable to the particular detector reagent may be observed on thesurface of the membrane. Optional wash steps may be added at any time inthe process, for instance, following application of the sample, and/orfollowing application of a detector reagent.

C. Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateralflow device is an analytical device having as its essence a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte mayflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; 6,258,548;6,699,722; and 6,368,876; EP 0810436; and WO 92/12428; WO 94/01775; WO95/16207; and WO 97/06439, each of which is incorporated by reference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials may be used, and rendered bibulous, e.g., byapplying a surfactant to the material), and allowed to migrate along thestrip until the liquid comes into contact with a specific bindingpartner (such as a lectin or antibody) that interacts with an analyte(such as one or more glycoprotein, glycolipid, or particular protein) inthe liquid. Once the analyte interacts with the binding partner, asignal (such as a fluorescent or otherwise visible dye) indicates thatthe interaction has occurred. Multiple discrete binding partners (suchas lectins or antibodies) may be placed on the strip (for example inparallel lines) to detect multiple analytes (such as glycoproteins,glycolipids, or particular proteins) in the liquid. The test strips mayalso incorporate control indicators, which provide a signal that thetest has adequately been performed, even if a positive signal indicatingthe presence (or absence) of an analyte is not seen on the strip.

The construction and design of lateral flow devices is very well knownin the art, as described, for example, in Millipore Corporation, A ShortGuide Developing Immunochromatographic Test Strips, 2nd Edition, pp.1-40, 1999, available by request at (800) 645-5476; and Schleicher &Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp.73-98, 2003, 2003, available by request at Schleicher & SchuellBioScience, Inc., 10 Optical Avenue, Keene, N.H. 03431, (603) 352-3810;both of which are incorporated herein by reference.

Lateral flow devices have a wide variety of physical formats that areequally well known in the art. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure.

The basic components of a particular embodiment of a lateral flow deviceare illustrated in FIG. 1A, which shows a particular embodiment of abibulous lateral flow strip 12. Lateral flow strip 12 is divided into aproximal sample application pad 14, an intermediate test result zone 16,and a distal absorbent pad 18. Flow strip 12 is interrupted by aconjugate pad 19 that contains labeled conjugate (such as gold- orlatex-conjugated lectin, antibody, or glycoprotein). A flow path alongstrip 12 passes from proximal pad 14, through conjugate pad 19, intotest result zone 16, for eventual collection in absorbent pad 18.Selective binding agents are positioned on a proximal test line 20 intest result membrane 16. A control line 22 is provided in test resultzone 16, slightly distal to test line 20. FIG. 1B illustrates a housing10 for test strip 12 that includes a rubber syringe dam 24 distal to theproximal sample application pad 14, a sufficiency indicator guide 26, avolumetric indicator 28 distal to the guide, a results window 30, and angrip area 32.

In operation of the particular embodiment of a lateral flow deviceillustrated in FIG. 1A, a fluid sample containing an analyte ofinterest, such as one or more glycoprotein, glycolipid, or particularprotein of interest, is applied to the sample pad 14. In some examples,the sample may be applied to the sample pad 14 by dipping the end of thedevice containing the sample pad 14 into the sample (such as urine orsaliva) or by applying the sample directly onto the sample pad 14 (forexample by placing the sample pad 14 in the mouth of the subject). Inother examples where a sample is whole blood, an optional developerfluid is added to the blood sample to cause hemolysis of the red bloodcells and, in some cases, to make an appropriate dilution of the wholeblood sample.

From the sample pad 14, the sample passes, for instance by capillaryaction, to the conjugate pad 19. In the conjugate pad 19, the analyte ofinterest, such as a glycoprotein, glycolipid, or particular protein, maybind (or be bound by) a mobilized or mobilizable detector reagent, suchas a glycoprotein (for example fetuin or an affinity-purified lectinbinding sample fraction), a lectin (for example one or more of PHA-E,LEL, AAL, DSL, ConA, SNA, or MAL) or an antibody (such as antibody thatrecognizes A1AG or A1AT). For example, a glycoprotein analyte may bindto a labeled (e.g., gold-conjugated or colored latexparticle-conjugated) glycoprotein, lectin or antibody contained in theconjugate pad. The analyte complexed with the detector reagent maysubsequently flow to the test result zone 16 where the complex mayfurther interact with an analyte-specific binding partner (such as alectin, an antibody that binds the lectin, a glycoprotein or otherparticular protein, an anti-hapten antibody, or streptavidin), which isimmobilized at the proximal test line 20. In some examples, a lectincomplexed with a detector reagent (such as, gold-conjugated lectin orantibody, labeled (e.g., gold-conjugated) antibody may further bind tounlabeled, oxidized antibodies or lectins immobilized at the proximaltest line 20. The formation of a complex, which results from theaccumulation of the label (e.g., gold or colored latex) in the localizedregion of the proximal test line 20 is detected. The control line 22 maycontain an immobilized, detector-reagent-specific binding partner, whichmay bind the detector reagent in the presence or absence of the analyte.Such binding at the control line 22 indicates proper performance of thetest, even in the absence of the analyte of interest. The test resultsmay be visualized directly, or may measured using a reader (such as ascanner). The reader device may detect color or fluorescence from thereadout area (for example, the test line and/or control line).

In another embodiment of a lateral flow device, there may be a second(or third, fourth, or more) test line located parallel or perpendicular(or in any other spatial relationship) to test line 20 in test resultzone 16 (for example test lines 20 a, 20 b, and 20 c in FIG. 1C). Theoperation of this particular embodiment is similar to that described inthe immediately preceding paragraph with the additional considerationsthat (i) a second detector reagent specific for a second analyte, suchas another lectin or antibody, may also be contained in the conjugatepad, and (ii) the second test line will contain a second specificbinding partner having affinity for a second analyte, such as a secondglycoprotein or glycolipid or a particular protein in the sample.Similarly, if a third (or more) test line is included, the test linewill contain a third (or more) specific binding partner having affinityfor a third (or more) analyte.

Some of the materials that may be useful for the components of a lateralflow device are shown in Table 2. However, one of skill in the art willrecognize that the particular materials used in a particular lateralflow device will depend on a number of variables, including, forexample, the analyte to be detected, the sample volume, the desired flowrate and others, and may routinely select the useful materialsaccordingly.

TABLE 2 Exemplary lateral flow device component materials ComponentUseful Material Sample Pad Glass fiber Woven fibers Screen Non-wovenfibers Cellulosic filters Paper Conjugate Pad Glass fiber PolyesterPaper Surface modified polypropylene Membrane Nitrocellulose (includingpure nitrocellulose and modified nitrocellulose) Nitrocellulose directcast on polyester support Polyvinylidene fluoride Nylon Absorbent PadCellulosic filters Paper1. Sample Pad

The sample pad (such as sample pad 14 in FIG. 1A) is a component of alateral flow device that initially receives the sample, and may serve toremove particulates from the sample. Among the various materials thatmay be used to construct a sample pad (see Table 2), a cellulose samplepad may be beneficial if a large bed volume (e.g., 250 μl/cm²) is afactor in a particular application. Sample pads may be treated with oneor more release agents, such as buffers, salts, proteins, detergents,and surfactants. Such release agents may be useful, for example, topromote resolubilization of conjugate-pad constituents, and to blocknon-specific binding sites in other components of a lateral flow device,such as a nitrocellulose membrane. Representative release agentsinclude, for example, trehalose or glucose (1%-5%), PVP or PVA(0.5%-2%), Tween 20 or Triton X-100 (0.1%-1%), casein (1%-2%), SDS(0.02%-5%), and PEG (0.02%-5%).

2. Membrane and Application Solution

The types of membranes useful in a lateral flow device (such asnitrocellulose, nylon and PVDF), and considerations for applying acapture reagent to such membranes have been discussed previously.

3. Conjugate Pad

The conjugate pad (such as conjugate pad 19 in FIG. 1A) serves to, amongother things, hold a detector reagent. In some embodiments, a detectorreagent may be applied externally, for example, from a developer bottle,in which case a lateral flow device need not contain a conjugate pad(see, for example, U.S. Pat. No. 4,740,468).

Detector reagent(s) contained in a conjugate pad are typically releasedinto solution upon application of the test sample. A conjugate pad maybe treated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose. A mixture of two or more release agents may be usedin any given application. In a particular disclosed embodiment, thedetector reagent in conjugate pad 19 is gold-conjugated lectin orantibody or a labeled anti-glycoprotein or glycolipid antibody. Inanother embodiment, the detector reagent includes a lectin bindingprotein conjugated to a colored latex particle and an additional bindingpartner (such as biotin or BSA-digoxigenin).

4. Absorbent Pad

The use of an absorbent pad 18 in a lateral flow device is optional. Theabsorbent pad acts to increase the total volume of sample that entersthe device. This increased volume may be useful, for example, to washaway unbound analyte from the membrane. Any of a variety of materials isuseful to prepare an absorbent pad, see, for example, Table 2. In somedevice embodiments, an absorbent pad may be paper (i.e., cellulosicfibers). One of skill in the art may select a paper absorbent pad on thebasis of, for example, its thickness, compressibility,manufacturability, and uniformity of bed volume. The volume uptake of anabsorbent made may be adjusted by changing the dimensions (usually thelength) of an absorbent pad.

IX. Methods for Identifying Compounds that Selectively Block or Alter aPre-Diabetic or Diabetic Glycosylation Profile

The identification herein of altered glycosylation profiles indicativeof pre-diabetes, diabetes, and diabetic complications provides anopportunity to identify compounds that prevent such changes inglycosylation profile. Disclosed herein are methods for identifying acompound that selectively decreases or blocks one or more type ofglycosylation that is increased in pre-diabetes, diabetes, or diabeticcomplication, such as one or more type of glycosylation that binds tolectins, such as AAL, DSL, PHA-E, ConA, SNA, LEL, or a combination oftwo or more thereof.

In some examples, the methods include identifying a compound thatselectively binds to one or more type of glycosylation that is increasedin pre-diabetes, diabetes, or diabetic complication. In a particularexample, a sample with a glycosylation profile indicative ofpre-diabetes, diabetes (such as a urine or saliva sample from a subjecthaving pre-diabetes or diabetes) is contacted with a test compound. Thesample binding to one or more lectin indicative of pre-diabetes ordiabetes (such as AAL, DSL, PHA-E, ConA, SNA, LEL, or a combination oftwo or more thereof) is determined. A decrease in sample binding to oneor more lectin (such as at least a 10% decrease, for example 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% 90%, or 95% decrease) as compared to acontrol or reference indicates a compound that selectively binds to oneor more type of glycosylation that is increased in pre-diabetes,diabetes, or diabetic complication.

In some examples, a test compound may be administered to a test subject,such as a subject with pre-diabetes or diabetes (such as an animalmodel, for example, a mouse or rat model of diabetes) for a period oftime sufficient to decrease or block one or more type of glycosylationthat is increased in pre-diabetes, diabetes, or diabetic complication.One of skill in the art may determine the appropriate time period oftreatment for a particular compound. In general, the compound isadministered to the subject for at least about one day, two days, fivedays, one week, two weeks, one month, three months, six months, or more.The glycosylation profile of a sample (such as urine or saliva) from thesubject is determined, for example by determining sample binding to oneor more lectins. A compound may be considered to decrease or block oneor more type of glycosylation associated with pre-diabetes, diabetes, ordiabetic complication if it decreases sample binding to one or morelectin (for example, AAL, DSL, PHA-E, ConA, SNA, LEL, or a combinationof two or more thereof) by at least about 10% (such as about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 99%) as compared to asample from a subject that is not treated with the test compound.

In embodiments, a “compound” or “test compound” may be any substance orany combination of substances that is useful for achieving an end orresult. The compounds identified using the methods disclosed herein maybe of use for selectively altering a glycosylation profile associatedwith pre-diabetes or diabetes. Any compound that has potential (whetheror not ultimately realized) to affect glycosylation profile may betested using the methods of this disclosure.

Appropriate compounds may be contained in libraries, for example,synthetic or natural compounds in a combinatorial library. Numerouslibraries are commercially available or may be readily produced; meansfor random and directed synthesis of a wide variety of organic compoundsand biomolecules, including expression of randomized oligonucleotides,such as antisense oligonucleotides and oligopeptides, also are known.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or may be readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Such libraries are useful for the screening of a large numberof different compounds.

Additional test compounds include lectins (for example, AAL, DSL, PHA-E,ConA, SNA LEL, MAL, or ECL), glycosylation inhibitors (for example,N-butyldeoxynojirimycin, castanospermine, 1-deoxymannojirimycin,1-deoxynorjirimycin, swainsonine, tunicamycin), foods (such as foodscontaining lectins, for example legumes, such as red kidney beans,soybeans or peanuts), and food extracts (such as fruit or vegetableextracts).

In embodiments, compounds that may alter a glycosylation profile, suchas a compound that selectively decreases or blocks one or more type ofglycosylation that is increased in pre-diabetes, diabetes, or diabeticcomplication, may be administered in any suitable manner, preferablywith pharmaceutically acceptable carriers. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present disclosure.The pharmaceutically acceptable carriers useful in this disclosure areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of the compoundsherein disclosed.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration may include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

The present disclosure is illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Glycoproteome Profile in Pre-Diabetes and Diabetes

This example describes serum and salivary glycoprotein profiles incontrol, pre-diabetic, and diabetic subjects.

Methods

Total glycoproteins were enriched from 3 mg of serum or saliva proteins,using Q-proteome glycoprotein fractionation columns (Qiagen, Valencia,Calif.) made of lectins such as ConA, LCH, WGA, and AIL, with standardbuffers as provided by the manufacturer. The quantities of the recoveredglycoproteins were estimated with a Bio-Rad DC protein assay (Bio-RadLaboratories, Hercules, Calif.). Samples from five subjects in eachrepresentative group were pooled for purification and gelelectrophoresis.

Serum or saliva proteins (50 μg) were labeled with CyDye DIGE Fluorminimal dye (Amersham Biosciences, Piscataway, N.J.) at a concentrationof 100-400 pmol of dye/50 μg of protein. Samples were labeled with Cy3(patient), Cy5 (control), or Cy2 (reference, patient+control), and allthree labeled samples were multiplexed and resolved in one gel. Labeledproteins were purified by acetone precipitation, dissolved in IEF bufferand rehydrated onto a 24 or 13-cm IPG strip (pH 4-7) for 12 hours atroom temperature. The IPG strip was subjected to 1-dimensionalelectrophoresis at 65-70 kVhrs. The IPG strip was then equilibrated withDTT equilibration buffer and IAA equilibration buffer for 15 minutessequentially before second-dimensional SDS-PAGE analysis. The IPG stripwas then loaded on to an 8-16% SDS-PAGE gel and electrophoresisconducted at 80-90 V for 18 hours to resolve proteins in the seconddimension.

Gels were scanned in a Typhoon 9400 scanner (Amersham Biosciences) usingappropriate lasers and filters with PMT voltage between 550-600. Imagesin different channels (control vs. IFG, IGT, and T2DM) were overlaidusing pseudo-colors, and differences were visualized using ImageQuantsoftware (Amersham Biosciences). 2D-gel image analysis to determine thedifferentially abundant protein spots was performed using Phoretix 2Devolution, version 2005 (Non-Linear Dynamics Ltd).

Results

Two-dimensional difference gel electrophoresis (2D DIGE) analysis wasused to analyze serum glycoproteins in samples from patients with IGT(FIG. 2A), IFG (FIG. 2B), and T2DM at diagnosis (FIG. 2C) and T2DM aftertreatment (FIG. 2D). Equal quantities of labeled proteins were labeledand subjected to 2D DIGE. Distinct changes in the serum glycoproteomeprofile were observed in the samples. Some glycoprotein spots wereincreased in IGT, IFG, or T2DM samples compared to controls, while otherglycoprotein spots were decreased in IGT, IFG, or T2DM samples comparedto controls (FIG. 2).

Salivary glycoproteins were also analyzed by 2D DIGE in samples frompatients with IGT (FIG. 3A), IFG (FIG. 3B), T2DM (FIG. 3C), and T1DM(FIG. 3D). As with the serum samples, distinct changes in the salivaryglycoproteome profile were observed in the samples. Some glycoproteinspots were increased in IGT, IFG, or T2DM samples compared to controls,while other glycoprotein spots were decreased in IGT, IFG, or T2DMsamples compared to controls (FIG. 3). In addition, the analysis showedthat some glycoproteins increased in T1DM as compared to T2DM (FIG. 3).

Example 2 Urinary Glycosylation Profile in Pre-Diabetes and Diabetes

This example describes the glycosylation profile of urine samples fromnon-diabetic and pre-diabetic individuals.

Methods

Urine samples were resuspended in 100 mM carbonate-bicarbonate buffer,pH 9.6, coated onto Reactibind™ plates (Pierce, Rockford, Ill.), andincubated overnight at 4° C. On the following day, plates were washed inphosphate buffered saline/Tween (PBST) using a Tecan® microplate washerand incubated with either biotinylated AAL, Con A, DSL, ECL, GSL-2, HHL,LEL, LTL, MAL, PHA-E, SNA, or WL (Vector Labs, Burlingame, Calif.)diluted in phosphate buffered saline (PBS). Plates were washed with 1.65ml PBST and incubated with streptavidin-horseradish peroxidase (HRP)(Pierce, Rockford, Ill.) dissolved in PBS. Plates were developed with3,3′,5,5′ tetramethylbenzidine (TMB) substrate (Neogen Corporation,Lexington, Ky.) and quenched with 2N H₂SO₄. Plates were analyzed using aSpectramax® plus microplate reader (Molecular Devices Corporation,Sunnyvale, Calif.).

Results

A direct lectin ELISA platform was developed to determine changes inspecific glycosylation profiles in urine from individuals withpre-diabetes and diabetes. In this platform, urine samples were coatedonto an ELISA plate, and global changes in particular types ofglycosylation were assessed by probing with biotinylated plant lectins.In the current study, a screen of urine was performed using lectins.Preliminary direct lectin ELISAs were conducted on pooled urine samplesfrom control, pre-diabetes and diabetes individuals. Relatively weaksignals were observed for GSL-2 (GlcNAc-GlcNAc), HHL (polymannose), LTL(α-1,2 fucose), and VVL (O-linked GalNac). Strong signals were observedfor lectins that recognized lactosamine (Gal-β1,4 GlcNAc), α-1,3 orα-1,6 fucosylation, and terminal sialylation.

A panel of urines from control subjects and patients with pre-diabetesor diabetes was probed. Following normalization by proteinconcentration, a significantly greater amount of lectin binding wasdetected in both pre-diabetes and diabetes samples compared to controlsusing AAL, DSL, and PHA-E biotinylated lectins (Table 3 and FIG. 4). Asignificantly greater amount of binding by ConA and SNA was alsoobserved in diabetes samples compared to controls (Table 3). The otherlectins employed did not detect significant differences between control,pre-diabetic, and diabetic urine samples. However, increased binding byConA, SNA, and LEL was observed in pre-diabetes samples compared tocontrols, and increased LEL binding was observed in diabetes samplescompared to controls, although these increases were not statisticallysignificant.

TABLE 3 Lectin binding to control, pre-diabetic and diabetic urinesamples Study Group (n) Control Pre-diabetes Diabetes Lectin (arbitraryunits) (44-50) (49-57) (43-47) AAL Geometric mean (sd)  473 (3) 1011 (3)1063 (3) p-value¹ Referent  0.002  0.001 DSL Geometric mean (sd)²  472(3) 1085 (3) 1080 (3) p-value¹ Referent  0.003  0.005 PHA-E Geometricmean (sd) 12462 (4)  35118 (7)  38228 (7)  p-value¹ Referent 0.02 0.02ConA Geometric mean (sd)  3507 (59) 20231 (10) 53142 (11) p-value³Referent 0.24 0.02 SNA Geometric mean (sd)  2748 (18) 6150 (9) 26680(3)  p-value³ Referent 0.81 0.04 LEL Geometric mean (sd) 2812 (4) 7328(7) 7910 (6) p-value³ Referent 0.13 0.07 MAL Geometric mean (sd) 2171(3)  1742 (20) 3268 (7) p-value³ Referent 0.94 0.56 ECL Geometric mean(sd) 3930 (3) 5225 (9) 5738 (9) p-value³ Referent 0.89 0.72 ¹Linearregression using Generalized Linear Models and Dunnett's 2-sidedpost-hoc correction factor for multiple comparisons versus the control²Lectin concentration/1000 ³Linear regression using Generalized LinearModels and Dunnett's T3 post-hoc correction factor to control forunequal variance across study groups

Lectin binding by control, pre-diabetic, and diabetic urine samples wasalso analyzed as a ratio of lectin binding (Table 4). The ratio of SNAbinding to MAL binding was significantly increased in diabetes samplescompared to controls. The ratio of ConA binding to MAL binding was alsosignificantly increased in diabetes samples compared to controls. Theratio of ConA binding to AAL binding in both pre-diabetes and diabetessamples was increased compared to controls, although this did not reachstatistical significance.

TABLE 4 Lectin binding ratio in control, pre-diabetic and diabetic urinesamples Study Group (n) Control Pre-diabetes Diabetes Lectin Ratio(43-50) (44-57) (43-47) SNA/MAL Geometric mean (sd) 1.4 (25)  3.3 (56)18.9 (7)  p-value¹ Referent 0.77 0.001 ConA/MAL Geometric mean (sd) 1.8(54) 11.2 (49) 29.3 (20) p-value¹ Referent 0.17 0.003 ConA/AAL Geometricmean (sd) 17.9 (66)    89 (11)   92 (10) p-value¹ Referent 0.09 0.09¹Linear regression using Generalized Linear Models and Dunnett's T3post-hoc correction factor to control for unequal variance across studygroups

These data provide a glycosylation profile indicative of pre-diabetes.The profile includes significantly increased binding of the sample tothe lectins AAL, DSL, PHA-E, or a combination of two or more thereof. Incontrast the binding of the sample to ConA, SNA, LEL, MAL, and ECL wasnot significantly changed compared to the control. Thus, a profile foridentifying pre-diabetes may include increased binding of the sample toAAL, DSL, PHA-E or a combination of two or more thereof. The profile mayalso be expressed as a ranking by amount of lectin binding using theresults of a statistical test, such as in increasing order of p-value.Such a glycosylation profile in pre-diabetes may include increasedbinding to AAL>DSL>PHA-E>LEL>ConA>SNA>ECL>MAL. A glycosylation profileindicative of pre-diabetes may also include increased lectin bindingratios, for example expressed as a ranking of lectin binding ratio inincreasing order of p-value. Such a glycosylation profile inpre-diabetes may include increased ConA/AAL>ConA/MAL>SNA/MAL.

These data also provide a glycosylation profile indicative of diabetes.The profile may include increased binding of the sample to the lectinsAAL, DSL, PHA-E, ConA, SNA, or a combination of two or more thereof. Incontrast, the binding of the sample to LEL, MAL, and ECL was notsignificantly changed compared to the control. Thus, a profile foridentifying diabetes may include increased binding of the sample to AAL,DSL, PHA-E, ConA, SNA, or a combination of two or more thereof. Theprofile may also be expressed as a ranking by amount of lectin bindingusing the results of a statistical test, such as in increasing order ofp-value. Such a glycosylation profile in diabetes may include increasedbinding to AAL>DSL>PHA-E=ConA>SNA>LEL>ECL>MAL. A glycosylation profileindicative of diabetes may also include increased lectin binding ratios,such as increased SNA/MAL, ConA/MAL, or a combination thereof. Theglycosylation profile of diabetes using lectin binding ratios may alsobe expressed using the results of a statistical test, such as inincreasing order of p-value. Such a glycosylation profile in diabetesmay include increased SNA/MAL>ConA/MAL>ConA/AAL ratios.

Example 3 Glycosylation of Alpha-1 Acid Glycoprotein in Pre-Diabetes andDiabetes

This example describes the glycosylation pattern (amount and type ofglycosylation) of a specific urinary protein in individuals withpre-diabetes and diabetes.

Methods

Concentrations of A1AG in urine were estimated by sandwich ELISA (Clarkand Adams, J. Gen. Virol. 34:475-483, 1977; Nerurkar et al., J. Clin.Microbiol. 20:109-114, 1984). A1AG polyclonal antibody (Dako,Carpinteria, Calif.; cat#A0011) was prepared in 100 mMcarbonate-bicarbonate buffer, pH 9.6, at a concentration of 2.0 μg/mland coated on a Reactibind™ plate by incubating overnight at 4° C. Theplate was then washed with PBST and blocked with 3% bovine serum albumin(BSA) in PBS for 1.5 hours at room temperature. After washing the plateswith PBST, appropriate dilutions of pure A1AG protein standard (Sigma,St. Louis, Mo.) and urine samples were added to the plate in triplicateand incubated for 1 hour. The plates were incubated with appropriatedilution of biotin-conjugated A1AG Dako polyclonal secondary antibodyfor 1 hour, and washed with 1.65 ml of PBST. Streptavidin-HRP conjugatewas added at a concentration of 0.1 mg/ml and incubated for 45 min priorto washing with PBST. TMB liquid substrate was then added followed byincubation at room temperature for 5-15 min for color development. Thereaction was stopped by the addition of 100 μl of 2N H₂SO₄. Absorbanceat 450 nm was measured on a Spectramax® Plus microplate reader(Molecular Devices Corporation, Sunnyvale, Calif.). A standard curve wasgenerated by either log-log or four-parameter curve fitting using theSoftmax® Pro v1.11 software (Molecular Devices Corporation, Sunnyvale,Calif.). The concentrations of the individual samples were estimatedfrom the average values of triplicates, in comparison to the standardcurve.

A1AG glycosylation pattern was determined using an antibody-lectinELISA. A1AG polyclonal antibody was coated onto a Reactibind™ plate asdescribed above. Following blocking with 3% BSA in PBS, the plates weretreated with 100 mM sodium metaperiodate, 50 mM citric acid, pH 4 for 15minutes. Urine samples were added to the plate and incubated for 3 hoursat room temperature. Following washing with PBST, biotin-conjugatedlectin was added and the plate was incubated at room temperature for 2hours. Development of the plate was also conducted as described inExample 1.

Results

It was first determined by protein ELISA that A1AG was present in urinein all the pre-diabetic and diabetic conditions. The amount of A1AG inthe pre-diabetic IGT and diabetic (NDM) urine was significantly higherthan in controls or IFG (Table 5).

The specific types of glycosylation of A1AG were probed with lectins bycapturing A1AG on the assay plate and probing the respective sugars withbiotinylated lectins (antibody-lectin ELISA; Table 5). A marginalincrease in AAL reactivity on A1AG in urine from IGT subjects wasobserved. There was a significant increase in the ConA reactivity ofA1AG in IGT urine compared to control. Lastly, there was a significantdifference in the amount of SNA-rective A1AG in NDM urine. Increases inCon A reactivity reflect a greater amount of biantennary content ofA1AG, and increases in AAL reactivity indicate a greater amount ofterminal fucose.

TABLE 5 A1AG protein and lectin binding to A1AG protein from urinesamples Control IFG IGT NDM (n = 42) (n = 16) (n = 27) (n = 24) IGT vs.control NDM vs. control Analyte Mean ± SD Mean ± SD Mean ± SD Mean ± SDp-value AUROC p-value AUROC A1AG  20.40 ± 6.12  32.32 ± 5.17  54.71 ±4.69  64.70 ± 2.12 0.0379 0.669 0.0103 0.723 A1AG + 325.47 ± 6.67 597.11± 9.47 855.92 ± 4.77 331.94 ± 5.71 0.0455 0.612 0.9663 0.528 ConA A1AG +678.81 ± 2.84 631.41 ± 9.29 777.09 ± 8.24 510.57 ± 2.92 0.7242 0.5470.2916 0.590 DSL A1AG + 445.96 ± 2.72 604.07 ± 2.21 769.75 ± 3.25 534.77± 2.76 0.0566 0.613 0.4769 0.526 AAL A1AG +  80.13 ± 59.91  255.36 ±50.44  440.01 ± 39.64 1429.25 ± 13.28 0.1000 0.626 0.0088 0.743 SNAA1AG + 440.60 ± 7.40 880.61 ± 3.85 977.37 ± 5.09  283.06 ± 10.04 0.10990.567 0.4144 0.589 MAL

These data provide glycosylation patterns of A1AG in pre-diabetes anddiabetes. A glycosylation pattern of A1AG in pre-diabetes may includeincreased binding of A1AG to the lectins ConA, AAL, or a combinationthereof. A glycosylation pattern of A1AG in diabetes may includeincreased binding of A1AG to the lectin SNA. The A1AG glycosylationpattern may also be expressed as a ranking by amount of lectin bindingusing the results of a statistical test, such as in increasing order ofp-value. Such an A1AG glycosylation pattern in pre-diabetes may includebinding to ConA>AAL>MAL>SNA>DSL. An A1AG glycosylation pattern indiabetes may include increased binding to SNA>DSL>MAL>AAL>ConA.

Example 4 Glycosylation Profile in Diabetic Complications

This example describes the glycosylation profile of urine samples fromindividuals with diabetic complications, such as chronic renal failure.

Methods

The glycosylation profile of urinary proteins was determined by lectinELISA, as described in Example 2. Concentrations of A1AG and A1AT inurine were estimated by sandwich ELISA as described in Example 3.

Results

A panel of urines from control subjects and patients with T2DM or T2DMwith chronic renal failure (T2DM/renal failure) were probed. The amountof both A1AG and A1AT in both T2DM and T2DM/renal failure urine wassignificantly higher than in controls (Table 6). A significantly greateramount of lectin binding was detected in both T2DM and T2DM/renalfailure samples compared to controls using AAL biotinylated lectin(Table 6). T2DM/renal failure samples also had significantly increasedSNA binding compared to controls; however, T2DM samples did not haveincreased SNA binding compared to controls (Table 6). ConA did notdetect significant differences between control, T2DM, and T2DM/renalfailure urine samples. The amount of A1AG and A1AT protein, as well asAAL and SNA reactivity was also higher in T2DM/renal failure samplesthan in T2DM samples.

TABLE 6 Lectin binding and protein expression in T2DM and renal failureControl T2DM/Renal (n = 10)* T2DM (n = 20)* Failure (n = 9)* A1AGGeometric Mean (SD) 24 (2) 137 (8)  7280 (2) P value¹ Referent 0.01<0.001 A1AT Geometric Mean (SD)  7 (3) 109 (11) 7500 (4) P value¹Referent  0.002 <0.001 AAL Geometric Mean (SD) 376 (2)  774 (2)  9197(2) P value² Referent 0.03 <0.001 SNA Geometric Mean (SD)³ 39 (2) 70 (3)2295 (3) P value¹ Referent 0.17 <0.001 ConA Geometric Mean (SD)³ 234(7)  93 (4) 1570 (3) P value¹ Referent 0.61 0.13 *Protein (ng/ml)¹Multiple comparisons against the control using Dunnett's T3 post-hoccorrection factor to account for unequal variance ²2-sided Dunnett'spost-hoc correction factor versus the control ³Concentration/10,000

These data provide glycosylation profiles for diabetes and the diabeticcomplication of renal failure. A glycosylation profile in diabetesincludes increased binding of the sample to the lectin AAL. Aglycosylation profile of a diabetic complication may include increasedbinding of the sample to the lectins AAL and SNA, or a combinationthereof. The glycosylation pattern may also be expressed as a ranking byamount of lectin binding using the results of a statistical test, suchas in increasing order of p-value. Such glycosylation profile indiabetes may include binding to AAL>>SNA>ConA. A glycosylation profilein diabetic complication may include increased binding to AAL=SNA>ConA.

Example 5 Exemplary Diagnostic Study

In embodiments, a saliva or urine sample may be obtained from subjectssuspected to have pre-diabetes (such as having an OGTT two-hour plasmaglucose of greater than or equal to 140 mg/dL and less than 200 mg/dL(7.8-11.0 mM) and/or a fasting plasma glucose (FPG) of greater than 100mg/dL and less than 126 mg/dL (5.6-6.9 mM)). A lectin ELISA may beperformed on the sample, and a glycosylation profile (as defined bybinding to one or more particular lectins, such as PHA-E, LEL, DSL, AAL,SNA, ConA, or MAL) may be determined relative to a glycosylation profilein a sample from a control subject without diabetes. A protein ELISA maybe performed on the sample, and the amount of A1AG and/or A1AT may bedetermined relative to the amount of these proteins in a sample from thecontrol.

An analysis may be performed, and the glycosylation profile may bealtered compared to the glycosylation profile of the sample from thenormal subject. An increase in one or more types of glycosylation in thesample from the subject may identify the subject as pre-diabetic. Theglycosylation profile may also be determined relative to theglycosylation profile in a sample from a subject known to be diabetic.An analysis may be performed, and the glycosylation profile may bealtered compared to the glycosylation profile of the sample from thediabetic subject. This may confirm that the subject is pre-diabetic.

An analysis may be performed, and the amount of A1AG and/or A1AT may bealtered compared to the amount of A1AG and/or A1AT in the sample fromthe normal subject. An increase in the amount of A1AG and/or A1AT in thesample from the subject may identify the subject as pre-diabetic. Theamount of A1AG and/or A1AT may also be determined relative to the amountof A1AG and/or A1AT in a sample from a subject known to be diabetic. Ananalysis may be performed, and the amount of A1AG and/or A1AT is alteredcompared to the amount of A1AG and/or A1AT in the sample from thediabetic subject. This may confirm that the subject is pre-diabetic.

Example 6 Exemplary Lateral Flow Device Diagnostic Test

FIG. 5 schematically illustrates an exemplary lateral flow device fordiagnosis of pre-diabetes or diabetes, including a test line forpre-diabetes or diabetes and a reference line. The device may detectbinding of glycoproteins in a sample to specific glycoprotein-bindingmolecules using a competitive assay format including a test lectin (TL)and a reference lectin (RL).

In the exemplary device, the conjugate pad 19 may include a test lectinbinding protein (TLBP) conjugate 40 (such as the TLBP fetuin covalentlyattached to blue latex particles and biotin) and a reference lectinbinding protein (RLBP) conjugate 42 (such as a protein fraction that isaffinity absorbed for TL binding and affinity purified for RL bindingcovalently attached to blue latex and BSA-digoxigenin). The TL (such asAAL) may be immobilized on the nitrocellulose membrane distal to theconjugate pad at the TL capture line 44. The RL (such as MAL) may beimmobilized on the nitrocellulose membrane distal to the TL test line 20at the RL capture line 46. In embodiments, the TL test line 20 may bestreptavidin and the RL test line 22 may be anti-digoxigenin.

The test may be performed by applying a sample (such as urine or saliva)from a subject to the sample application pad 14. The sample may flowthrough the conjugate pad 19, releasing the TLBP conjugate and the RLBPconjugate. The displaced TLBP conjugate may be captured by the TL testline 20. The displaced RLBP conjugate may be captured by the RL testline 22. The TL test line intensity may be compared to the RL test lineintensity (for example, visually or using a reader). If the TL test lineintensity is greater than the RL test line intensity, then the subjectmay be diagnosed as normal. If the TL test line intensity is less thanthe RL test line intensity, then the subject may be diagnosed as havingpre-diabetes or diabetes.

Although certain embodiments have been illustrated and described herein,it will be appreciated by those of ordinary skill in the art that a widevariety of alternate and/or equivalent embodiments or implementationscalculated to achieve the same purposes may be substituted for theembodiments shown and described without departing from the scope. Thosewith skill in the art will readily appreciate that embodiments may beimplemented in a very wide variety of ways. This application is intendedto cover any adaptations or variations of the embodiments discussedherein. Therefore, it is manifestly intended that embodiments be limitedonly by the claims and the equivalents thereof.

What is claimed is:
 1. A method for identifying a subject withpre-diabetes or diabetes, comprising: determining a first glycosylationprofile of a first sample from the subject, wherein the firstglycosylation profile comprises one or more parameters relating toglycosylation; and comparing the first glycosylation profile to a secondglycosylation profile of a second sample from a control subject, whereinthe second glycosylation profile comprises one or more parametersrelating to glycosylation, wherein the control subject does not havepre-diabetes or diabetes; wherein determining the glycosylation profileof the first and second samples comprises measuring binding of Aleuriaaurantia lectin, Datura stramonium lectin, Phaseolus vulgaris agglutininlectin, Concanavalin A, and Sambucus nigra lectin to the first andsecond samples; and wherein an increase in binding of Aleuria aurantialectin, Datura stramonium lectin, and Phaseolus vulgaris agglutininlectin to the first sample relative to the second sample indicates thatthe subject has either pre-diabetes or diabetes; and wherein an increasein binding of Aleuria aurantia lectin, Datura stramonium lectin,Phaseolus vulgaris agglutinin lectin, Concanavalin A, and Sambucus nigralectin to the first sample relative to the second sample indicates thatthe subject has diabetes, and not pre-diabetes.
 2. The method of claim1, wherein both of the first and second samples comprise urine, saliva,or serum.
 3. The method of claim 1, wherein an increase in ConcanavalinA and/or Sambucus nigra lectin binding to the first sample relative tothe second sample indicates the subject has diabetes.
 4. The method ofclaim 1, further comprising determining a quantity of alpha-1 acidglycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in thesample from the subject, wherein an increase in the quantity of thealpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT)protein in the sample from the subject as compared to the sample fromthe control subject indicates that the subject has pre-diabetes ordiabetes.
 5. The method of claim 4, wherein determining the quantity ofthe alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT)protein comprises performing an immunoassay.
 6. The method of claim 1,further comprising: determining a glycosylation state of alpha-1 acidglycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in thefirst sample; and comparing the glycosylation state of the alpha-1 acidglycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in thefirst sample to the glycosylation state of the alpha-1 acid glycoprotein(A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the second sample,wherein a difference in the glycosylation state of the alpha-1 acidglycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in thefirst sample as compared to the glycosylation state of the alpha-1 acidglycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in thesecond sample indicates that the subject has pre-diabetes or diabetes.7. The method of claim 6, wherein determining the glycosylation state ofthe alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT)protein comprises detecting and/or measuring lectin binding to thealpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT)protein.
 8. The method of claim 7, wherein determining the glycosylationprofile of the first and second samples further comprises measuringbinding of Concanavalin A and Sambucus nigra lectin: and wherein one ormore of the Aleuria aurantia lectin, Datura stramonium lectin, Phaseolusvulgaris agglutinin lectin, Concanavalin A, or Sambucus nigra lectinbind alpha-1 acid glycoprotein.
 9. The method of claim 6, furthercomprising determining a quantity of the alpha-1 acid glycoprotein(A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the first and secondsamples.
 10. The method of claim 9, wherein an increase in the quantityof alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) inthe first sample as compared to the second sample indicates that thesubject has pre-diabetes or diabetes.
 11. The method of claim 9, whereindetermining the quantity of the alpha-1 acid glycoprotein (A1AG) and/oralpha-1 antitrypsin (A1AT) protein comprises performing an immunoassay.12. The method of claim 1, wherein determining the glycosylation profileof the first sample comprises contacting a the first sample with alateral flow device comprising the Aleuria aurantia lectin, Daturastramonium lectin, and Phaseolus vulgaris agglutinin lectin.
 13. Themethod of claim 1, wherein an increase in blinding of the Aleuriaaurantia lectin, Datura stramonium lectin, or Phaseolus vulgarisagglutinin lectin to the first sample relative to the second sampleindicates that the subject has a diabetic complication.
 14. The methodof claim 13, wherein the diabetic complication is a microvascularcomplication or a macrovascular complication.
 15. The method of claim14, wherein the diabetic complication is a microvascular complicationcomprising one or more of diabetic nephropathy, diabetic retinopathy,and diabetic neuropathy.